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pPICZα A, B, and C - Thermo Fisher Scientific

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1. 2 5 units ul 1 ul Incubate at 16 C for 2 5 hours Heat inactivate the ligase by incubating at 65 C for 20 minutes Add the following reagents for restriction enzyme digestion cut back Note that BamHI and BglII may be used with the same reaction buffer Sterile water 23 pl 10X restriction enzyme buffer 5 pl BglII 10 units pl 1 ul BamHI 10 units pl 1 ul Incubate the reaction at 37 C for 2 hours Add 50 pl of phenol chloroform and extract the restriction enzyme digestion to remove the enzymes Transfer the aqueous solution to a new microcentrifuge tube 7 Toethanol precipitate the DNA add 5 pl of 3 M sodium acetate and 110 pl of 100 ethanol 8 Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant 9 Wash the nucleic acid pellet with 80 ethanol centrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol and air dry the pellet 10 Resuspend pellet in 4 ul sterile water Save on ice if you plan to ligate your insert immediately or you can store at 20 C Proceed to Ligation of Multimers into Linearized Vector next page Continued on next page 29 Construction of n Vitro Multimers continued You may wish to combine the ligation reaction with the restriction enzyme digestion to enrich for head to tail multimers Use the reaction buffer for the Note Se SES restriction enzymes and add 1 mM ATP to the reaction in or
2. Coit D G Heberlein U A Masiarz G R Mullenbach G T Urdea M S Valenzuela P and Barr P J 1984 a Factor Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae Proc Natl Acad Sci USA 81 4642 4646 Calmels T Parriche M Burand H and Tiraby G 1991 High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance Curr Genet 20 309 314 Drocourt D Calmels T P G Reynes J P Baron M and Tiraby G 1990 Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance Nuc Acids Res 18 4009 Ellis S B Brust P F Koutz P J Waters A F Harpold M M and Gingeras T R 1985 Isolation of Alcohol Oxidase and Two other Methanol Regulatable Genes from the Yeast Pichia pastoris Mol Cell Biol 5 1111 1121 Evans G I Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies Specific for c myc Proto oncogene Product Mol Cell Biol 5 3610 3616 Gietz R D and Schiestl R H 1996 Transformation of Lithium Treated Yeast Cells and the Selection of Auxotrophic and Dominant Markers In Methods in Molecular Biology I H Evans ed Totowa NJ Humana Press Henikoff S and Cohen E H 1984 Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces cerevisiae Mol Cell Biol 4 151
3. Ligate into BamHI linearized recombinant pPICZa Transform into E coli and analyze recombinant plasmids for copy number by digesting with Bg II and BamHI Alternative You may wish to build each desired multimer in increments by ligating each Procedure additional expression cassette one or two at a time into pPICZa A B or C For example Stage Description 1 Digest pPICZa containing one copy of your gene with BamHI 2 Ligate a single copy of the BglII BamHI expression cassette into BamHI digested vector 3 Transform E coli and analyze the transformants for the vector with 2 copies of your insert 4 Isolate and digest this vector with 2 copies of your gene with BamHI and Bglll to release a cassette with 2 copies of your gene optional 5 Digest the vector with 2 copies of your gene with BamHI and ligate 1 or 2 copies see Step 4 of the expression cassette into the vector 6 Transform E coli and analyze the transformants for the vector with 3 or 4 copies of your insert 7 Repeat until the desired multimer is reached Continued on next page 25 Construction of n Vitro Multimers continued Before Starting You will need to have the following materials on hand e Electrocompetent or chemically competent E coli must be recA endA for transformation You will need 3 4 tubes of competent cells per experiment e BamHI and BglII restriction enzymes and appropriate buffers e Low melt agarose e S N A P Gel P
4. Sample Application Native Conditions Sample Application Denaturing Conditions Note Analysis of Purification Scale up 16 The following protocol may be used for chromatography of medium For sample application onto ProBond you will need Native Binding Buffer pH 7 8 and a 2 ml ProBond column pre equilibrated using native conditions 1 Combine 1 ml of medium with 7 ml Native Binding Buffer 2 Take a pre equilibrated ProBond column and resuspend the resin in 4 ml of the diluted lysate from Step 1 3 Seal the column and batch bind by rocking gently at room temperature for 10 minutes 4 Let the resin settle by gravity or low speed centrifugation 800 x g and carefully remove the supernatant Save the supernatant to check for unbound protein 5 Repeat Steps 2 through 4 with the remaining 4 ml of diluted lysate Proceed to Column Washing and Elution Under Native Conditions in the ProBond Purification manual Use the recommendations noted for bacterial cell lysates Use the protocol above except pre equilibrate the ProBond column using Denaturing Binding Buffer and combine 1 ml of the Pichia cell lysate with 7 ml of the Denaturing Binding Buffer We have observed that some Pichia proteins may be retained on the ProBond column using native purification conditions Optimization of the purification see ProBond Purification manual or using denaturing purification may remove these non s
5. T and Hashimoto Gotoh T 1988 Tandem Gene Amplification in vitro for Rapid and Efficient Expression in Animal Cells Gene 71 9 18 Taylor W H and Hagerman P J 1987 A General Method for Cloning DNA Fragments in Multiple Copies Gene 53 139 144 32 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Certificate of Analysis Limited Warranty Safety Data Sheets SDSs are available at www invitrogen com sds The Certifi
6. The number of copies you obtain may depend on how well a large vector is tolerated by the host strain 4 Once you have identified plasmids with multiple copies of your expression cassette be sure to purify by streaking for single colonies and confirming your construct 5 Prepare frozen glycerol stocks of E coli containing each of your multimeric constructs 6 Prepare at least 100 ug of each plasmid for transformation into Pichia You need more DNA because you will be transforming with uncut plasmid DNA Transformation efficiency is about 1 to 2 orders of magnitude less for uncut versus linearized DNA 7 Proceed to Pichia Transformation page 9 Use optional overgrowth step in the procedure on page 12 Continued on next page 31 Construction of n Vitro Multimers continued Troubleshooting The table below will help you optimize formation and isolation of multimers in Pichia Problem Possible Reason Solution No multimers or low number of CIAP defective Use fresh CIAP multimers in your vector after transformation into E coli Add more CIAP Add 1 unit of CIAP and incubate 15 more minutes at 37 C This is somewhat risky as CIAP can degrade the ends of your DNA Not enough insert DNA to ligate Add more BamHlI BgIll expression cassette to your ligation Construct is unstable in E coli Decrease the number of cassettes in the vector Multimers are too long to ligate efficiently Try l
7. Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cycle at 15 psi and 121 C for 20 minutes 3 Allow the medium to cool to at least 55 C before adding the Zeocin to 25 ug ml final concentration Store plates at 4 C in the dark Plates containing Zeocin are stable for up to 2 weeks Yeast Extract Peptone Dextrose Medium 1 liter 1 yeast extract 2 peptone 2 dextrose glucose 2 agar appropriate concentration of Zeocin 1 Dissolve 10 g yeast extract 20 g of peptone in 900 ml of water Include 20 g of agar if making YPD slants or plates Autoclave for 20 minutes on liquid cycle Add 100 ml of 20 dextrose filter sterilize dextrose before use TM Cool solution to 60 C and add the appropriate amount of Zeocin from a 100 mg ml stock solution Che es Note It is necessary to include Zeocin in the medium for selection of Pichia transformants only Zeocin may be omitted from the medium when performing expression studies Store YPD slants or plates containing Zeocin at 4 C The shelf life is one to two weeks Continued on next page 17 Recipes continued YPDS Zeocin Agar 18 Yeast Extract Peptone Dextrose Medium with Sorbitol 1 liter 1 yeast extract 2 peptone 2 dextrose glucose 1 M sorbitol 2 agar appropriate concentration of Zeocin 1 Dissolve 10 g yeast extract 182 2 g sorbitol 20 g of peptone i
8. CAT CAT CAT CAT CAT CAT TGA GTTTGTA Glu Asp Leu Asn Ser Ala Val Asp His His His His His His GCCTTAGACA TGACTGTTCC TCAGTTCAAG TTGGGCACTT ACGAGAAGAC CGGTCTTGCT 3 AOX7 priming site AGATTCTAAT CAAGAGGATG TCAGAATGCC ATTTGCCTGA GAGATGCAGG CTTCATTTTT 3 polyadenylation site GATACTTTTT TATTTGTAAC CTATATAGTA TAGGATTTTT TTTGTCATTT TGTTTCTTCT To express your protein with a native N terminus you must use PCR and utilize the Xho I site upstream of the Kex2 cleavage site to clone your gene flush with the Kex2 cleavage site see page 4 for more details Continued on next page Cloning into pPICZa A B and C continued Multiple Cloning Below is the multiple cloning site for pPICZa C Restriction sites are labeled to Site of pPICZa C indicate the cleavage site The boxed nucleotides indicate the variable region 811 871 931 983 1034 1085 1136 1187 1244 1301 1353 1413 1473 The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pPICZa C is available for downloading at www invitrogen com or from Technical Support see page 33 For a map and a description of the features of pPICZa refer to pages 21 22 5 end of AOX1 mRNA 5 AOX7 priming site AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA al CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG ATG AGA TTT CCT TCA ATT TTT ACT G
9. Inc Tucson Arizona Life Technologies has an exclusive license to sell Pichia expression kits and vectors to scientists for research purposes only under the terms described below Use of Pichia pastoris by commercial entities for any commercial purpose requires the user to obtain a commercial license as detailed below Before using any Pichia expression product please read the following license agreement If you do not agree to be bound by its terms contact Life Technologies within 10 days for authorization to return the unused Pichia expression products and to receive a full refund If you do agree to the terms of this license agreement please complete the User Registration Card and return it to Life Technologies before using the product Life Technologies Corporation Life Technologies grants you a non exclusive license to use the enclosed Pichia expression vectors Expression Vector for academic research or for evaluation purposes only The Expression Vectors are being transferred to you in furtherance of and reliance on such license You may not use the Expression Vectors for any commercial purpose without a license for such purpose from Research Corporation Technologies Inc Tucson Arizona Commercial purposes include any use of Expression Products or Expression Vectors in a Commercial Product any use of Expression Products or Expression Vectors in the manufacture of a Commercial Product any sale of Expression Products any use of E
10. Site of pPICZa A indicate the cleavage site The boxed nucleotides indicate the variable region 811 871 931 983 1034 1085 1136 1187 244 299 351 411 471 The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pPICZa A is available for downloading at www invitrogen com or from Technical Support see page 33 For a map and a description of the features of pPICZa refer to pages 21 22 D end of AOX1 mRNA 5 AOX1 priming site p AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA 1 CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA GCA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala a factor signal sequence CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG TTT Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Xho it T ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA TCT CTC Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Kex2 signal cleavage EcoRI pm Stii BsmB Asp718 GA
11. cerevisiae as suggested by the manufacturer of the specific electroporation device being used Immediately add 1 ml of ice cold 1 M sorbitol to the cuvette Transfer the cuvette contents to a sterile 15 ml tube Let the tube incubate at 30 C without shaking for 1 to 2 hours Spread 50 200 ul each on separate labeled YPDS plates containing the appropriate concentration of Zeocin Incubate plates for 2 to 3 days at 30 C until colonies form Pick 10 20 colonies and purify streak for single colonies on fresh YPD or TM YPDS plates containing the appropriate concentration of Zeocin Continued on next page 11 Pichia Transformation continued Note Mut Phenotype 12 Generally several hundred Zeocin resistant colonies are generated using the protocol on the previous page If more colonies are needed the protocol may be modified as described below Note that you will need 20 150 mm plates with YPDS agar containing the appropriate concentration of Zeocin 1 Set up two transformations per construct and follow Steps 1 through 5 of the Transformation by Electroporation protocol page 11 2 After 1 hour in 1M sorbitol at 30 C Step 5 previous page add 1 ml YPD medium to each tube 3 Shake 200 rpm the cultures at 30 C After 1 hour take one of the tubes and plate out all of the cells by spreading TM 200 ul on 150 mm plates containing the appropriate concentration of Zeocin 5 Optional C
12. of detection does not reveal any expression you may want to consider using a more sensitive method Once a positive clone has been identified large scale expression can be carried out in shake flask or fermentation and expression conditions can be optimized Note that once you have obtained Zeocin resistant transformants it is not necessary to maintain your recombinant Pichia clone in medium containing TM TM Zeocin for expression studies Zeocin is only required for initial screening and selection of recombinant clones We recommend that you use the following techniques to assay expression of your protein Remember to analyze BOTH the medium and the cells for the presence of your recombinant protein Note that the o factor signal sequence will add approximately 9 3 kDa to the size of your protein if it is unprocessed The C terminal tag will add 2 5 kDa to the size of your protein Be sure to account for any additional amino acids that are in between the signal sequence processing sites and the N terminus of your protein and also the end of your protein and the C terminal tag Technique Method of Detection Sensitivity SDS PAGE Visualization by eye Can detect as little as 100 ng Coomassie stained in a single band SDS PAGE Visualization by eye Can detect as little as 2 ng in a Silver stained single band Western Analysis Antibody to your particular protein Can detect as little as 1 10 pg depending on detectio
13. of the recombinant fusion Protein protein using metal chelating resins such as ProBond Ordering information for ProBond resin is provided below Item Quantity Cat no ProBond Purification System 1 kit K850 01 ProBond Purification System 1 kit K852 01 with Anti myc HRP Antibody ProBond Purification System 1 kit K853 01 with Anti His C term HRP Antibody ProBond Resin 50 ml R801 01 150 ml R801 15 Purification Columns 50 polypropylene columns R640 50 viii Overview Introduction Experimental Overview Introduction pPICZa A B and C are 3 6 kb vectors used to express and secrete recombinant proteins in Pichia pastoris Recombinant proteins are expressed as fusions to an N terminal peptide encoding the Saccharomyces cerevisiae a factor secretion signal The vector allows high level methanol inducible expression of the gene of interest in Pichia and can be used in any Pichia strain including X 33 SMD1168H and KM71H pPICZa contains the following elements e D fragment containing the AOX1 promoter for tightly regulated methanol induced expression of the gene of interest Ellis et al 1985 Koutz et al 1989 Tschopp et al 1987a e a factor secretion signal for directing secreted expression of the recombinant protein e Zeocin resistance gene for selection in both E coli and Pichia Baron et al 1992 Drocourt et al 1990 e C terminal peptide containing
14. predicted protease cleavage sites for the a factor signal sequence are indicated in the diagrams on pages 5 7 The native 5 end of the AOX1 mRNA is noted in the diagram for each multiple cloning site This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason pPICZa is a terminal fusion vector To express your gene as a recombinant fusion protein you must clone your gene in frame with the N terminal o factor secretion signal and the C terminal peptide containing the c myc epitope and the polyhistidine tag The vector is supplied in three reading frames to facilitate cloning Refer to the diagrams on pages 5 7 to develop a cloning strategy Note The initiation ATG in the a factor signal sequence corresponds to the native initiation ATG of the AOX1 gene If you wish to express your protein without the C terminal peptide be sure to include a stop codon The processing of the a factor signal sequence in pPICZa occurs in two steps 1 The preliminary cleavage of the signal sequence by the KEX2 gene product with the final Kex2 cleavage occurring between arginine and glutamine in the sequence Glu Lys Arg Glu Ala Glu Ala where is the site of cleavage 2 The Glu Ala repeats are further cleaved by the STE13 gene product In Saccharomyces cerevisiae it has been noted that the Glu Ala repeats are not necessary for cleavage by Kex2 but cleavage after Glu Lys A
15. step below 2 Cut out the gel slice containing the PCR product and melt it at 65 C in 2 volumes of the 6 M sodium iodide solution 3 Add 1 5 volumes Binding Buffer Load solution no more than 1 ml at a time from Step 3 onto a S N A P column Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant TM If you have solution remaining from Step 3 repeat Step 4 Add 900 ul of the Final Wash Buffer Centrifuge 1 minute at full speed in a microcentrifuge and discard the flow through Repeat Step 7 Elute the purified DNA in 15 ul of sterile water Store on ice if proceeding immediately to Ligation of Expression Cassette next page Store at 20 C for long term storage Continued on next page 27 Construction of n Vitro Multimers continued Dephosphorylation Dephosphorylation of the BamHI digested vector is necessary to prevent self ligation of Vector 28 1 S ER in 10 Take your BamHl digest from Digestion of Recombinant pPICZa Step 2 and phenol extract then ethanol precipitate the DNA Resuspend in 17 ul of sterile water Set up a 20 ul dephosphorylation reaction in a microcentrifuge tube as follows BamHI digested recombinant pPICZa page 27 top Step 2 17 ul 10X CIAP Buffer 2 ul CIAP 1 Unit ul Lu Incubate at 37 C for 15 minutes Add 30 ul of sterile water to the reaction for a final volume of 50 ul Add 50 pl of phenol chloroform and extract your DNA soluti
16. the c myc epitope and a polyhistidine 6xHis tag for detection and purification of a recombinant fusion protein if desired Three reading frames to facilitate in frame cloning with the C terminal peptide The following table describes the basic steps needed to clone and express your gene of interest in pPICZa Step Action Page 1 Propagate pPICZa A B and C by transformation into a recA 2 endAl E coli strain such as TOP10 DH5a or JM109 2 Develop a cloning strategy and ligate your gene into one of the 3 7 pPICZa vectors in frame with the a factor secretion signal and the C terminal tag 3 Transform into E coli and select transformants on Low Salt LB 8 plates containing 25 ug ml Zeocin 4 Analyze 10 20 transformants by restriction mapping or 8 sequencing to confirm in frame fusion of your gene with the a factor secretion signal and the C terminal tag 5 Purify and linearize the recombinant plasmid for transformation 8 10 into Pichia pastoris 6 Transform your Pichia strain and plate onto YPDS plates 11 containing the appropriate concentration of Zeocin 7 Select for Zeocin resistant transformants 11 12 8 Optimize expression of your gene 13 14 9 Purify your fusion protein on metal chelating resin 15 16 Le ProBond Methods Cloning into pPICZa A B and C Introduction General Molecular Biology Techniques E coli Strain Tra
17. the solution to pellet the DNA wash the pellet with 80 ethanol air dry and resuspend in 10 pl sterile deionized water Use immediately or store at 20 C Continued on next page Pichia Transformation continued Preparing Pichia for Electroporation Transformation by Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at 30 C overnight Inoculate 500 ml of fresh medium in a 2 liter flask with 0 1 0 5 ml of the overnight culture Grow overnight again to an OD 1 3 1 5 Centrifuge the cells at 1500 x g for 5 minutes at 4 C Resuspend the pellet with 500 ml of ice cold 0 C sterile water Centrifuge the cells as in Step 3 then resuspend the pellet with 250 ml of ice cold 0 C sterile water Centrifuge the cells as in Step 3 then resuspend the pellet in 20 ml of ice cold 0 C 1 M sorbitol Centrifuge the cells as in Step 3 then resuspend the pellet in 1 ml of ice cold 1 M sorbitol for a final volume of approximately 1 5 ml Keep the cells on ice and use that day Do not store cells Mix 80 ul of the cells from Step 6 above with 5 10 pg of linearized pPICZa DNA in 5 10 ul sterile water and transfer them to an ice cold 0 C 0 2 cm electroporation cuvette Incubate the cuvette with the cells on ice for 5 minutes Pulse the cells according to the parameters for yeast Saccharomyces
18. with shaking to an OD of 0 8 to 1 0 approximately 108 cells ml 2 Harvest the cells wash with 25 ml of sterile water and centrifuge at 1500 x g for 10 minutes at room temperature 3 Resuspend the cell pellet in 1 ml of 100 mM LiCl and transfer the suspension to a 1 5 ml microcentrifuge tube 4 Pellet the cells at maximum speed for 15 seconds and remove the LiCl with a pipet 5 Resuspend the cells in 400 ul of 100 mM LiCl Dispense 50 ul of the cell suspension into a 1 5 ml microcentrifuge tube for each transformation and use immediately Do not store on ice or freeze at 20 C Continued on next page 23 Lithium Chloride Transformation Method continued Transformation 24 1 Bot Gl Boil a 1 ml sample of single stranded DNA for 5 minutes then quickly chill on ice Keep on ice Note It is neither necessary nor desirable to boil the carrier DNA prior to each use Store a small aliquot at 20 C and boil every 34 times the DNA is thawed Centrifuge the cells from Step 6 above and remove the LiCl with a pipet For each transformation add the following reagents in the order given to the cells PEG shields the cells from the detrimental effects of the high LiCl concentration 240 ul 50 PEG 36 ul 1 M LiCl 25 pl 2 mg ml single stranded DNA Plasmid DNA 5 10 ug in 50 ul sterile water Vortex each tube vigorously until the cell pellet is completely mixed 1 minute Incubate the tube at 30 C
19. 0 to 100 fold drop in transformation efficiency However with selection on Zeocin any transformants you obtain will probably contain your construct For best results e Use electroporation to transform your cells e Use at least 50 ug plasmid DNA for each transformation Plate out all of the transformation mix on several YPDS plates containing the appropriate concentration of Zeocin You may also use increasing concentrations of Zeocin to isolate multi copy transformants You will need to use the optional overgrowth step in the procedure on page 12 Digesting Set up two separate digests of recombinant pPICZa containing one copy of your Recombinant gene pPICZa 1 Double digest 1 2 ug of recombinant pPICZa in 20 ul with 10 units each of Bglll and BamHI Proceed to Production of Expression Cassettes for Multimerization Step 1 Digest 2 ug of recombinant pPICZa in 20 pl with 10 units of BamHI only 3 Proceed to Dephosphorylation of Vector Step 1 Producing The S N A P Gel Purification Kit available from Invitrogen see page vii allows Expression you to rapidly purify DNA fragments from regular agarose gels Alternatively Cassettes for you may use glass milk To use the S N A P Gel Purification Kit follow the Multimerization steps below 1 Electrophorese your BamHI BglII digest from Step1 above on a 1 to 5 regular TAE agarose gel Note Do not use TBE to prepare agarose gels Borate interferes with the sodium iodide
20. 5 1520 Higgins D R and Cregg J M 1998 Pichia Protocols In Methods in Molecular Biology Vol 103 J M Walker ed Humana Press Totowa NJ Irniger S Egli C M and Braus G H 1991 Different Classes of Polyadenylation Sites in the Yeast Saccharomyces cerevisiae Mol Cell Bio 11 3060 3069 Koutz P J Davis G R Stillman C Barringer K Cregg J M and Thill G 1989 Structural Comparison of the Pichia pastoris Alcohol Oxidase Genes Yeast 5 167 177 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Mulsant P Tiraby G Kallerhoff J and Perret J 1988 Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells Somat Cell Mol Genet 14 243 252 Perez P Tiraby G Kallerhoff J and Perret J 1989 Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation Plant Mol Biol 13 365 373 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Scorer C A Buckholz R G Clare J J and Romanos M A 1993 The Intracellular Production and Secretion of HIV 1 Envelope Protein in the Methylotrophic Yeast Pichia pastor
21. CT GTT TTA TTC GCA GCA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala a factor signal sequence CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG TTT Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Xho T ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA TCT CTC Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Kex2 signal cleavage Cal EcoRI Pmli Sfi 1 BsmB GAG AAG AGA GAG GCT GAA GC ATCGAT GAATTCAC GTGGCCCAG CCGGCCGTC TCGGA Glu Lys Arg Glu AlagGlu Alas Ste13 signal cleavage Asp718 Kpn Xho Sac Il Not Xba c myc epitope TCGGTACCTC GAGCCGCGGC GGCCGCCAGC TTTCTA GAA CAA AAA CTC ATC TCA GAA Glu Gln Lys Leu Ile Ser Glu polyhistidine tag I GAG GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTA Glu Asp Leu Asn Ser Ala Val Asp His His His His His His GCCTTAGACA TGACTGTTCC TCAGTTCAAG TTGGGCACTT ACGAGAAGAC CGGTCTTGCT 3 AOX7 priming site l AGATTCTAAT CAAGAGGATG TCAGAATGCC ATTTGCCTGA GAGATGCAGG CTTCATTTTT 3 polyadenylation site GATACTTTTT TATTTGTAAC CTATATAGTA TAGGATTTTT TITGTCATTT TGTTTCTTCT To express yo
22. G AAA AGA GAG GCT GAA sch GAATTCAC GTGGCCCAG CCGGCCGTC TCGGATCGGT Glu Lys Arg Glu AlagGlu Alag Ste13 signal cleavage Kpn 1 Xho Sac Il Not Xba c myc epitope ACCTCGAGCC GCGGCGGCC GCCAGCTTTC TA GAA CAA AAA CTC ATC TCA GAA GAG Glu Gln Lys Leu Ile Ser Glu Glu polyhistidine tag aan Pate GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC Asp Leu Asn Ser Ala Val Asp His His His His His His TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA 3 AOX7 priming site l TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT 3 polyadenylation site ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TICTTCTCGT To express your protein with a native N terminus you must use PCR and utilize the Xho I site upstream of the Kex2 cleavage site to clone your gene flush with the Kex2 cleavage site see page 4 for more details Continued on next page Cloning into pPICZa A B and C continued Multiple Cloning Below is the multiple cloning site for pPICZa B Restriction sites are labeled to Site of pPICZa B indicate the cleavage site The boxed nucleotides indicate the variable region 811 871 931 983 1034 1085 1136 1187 1243 1300 1392 1412 1472 The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pPICZa B is available for downloading at www invitrogen com or from Technical Sup
23. Invitrogen by technologies pPICZa A B and C Pichia expression vectors for selection on Zeocin and purification of secreted recombinant proteins Cat no V195 20 Rev Date 7 July 2010 Manual part no 25 0150 MANO0000035 ii Table of Contents Important Information giseagan inten ansioso on habe v Accessory Products css 2 ran a OI AUN vii Introduction sons so onssenneennsennanenanenansnsnsnnnsnnnsnneennnennnnenneenanennnsenanenaaennanenavenasenassnavsnnennnnennmenasesnssensvenseens 1 Overview lo elo dl 1 E UL EE 2 Cloning into pPIEZa A Band Einicio 2 Multiple Cloning Site of pPICZa A EEN 5 Multiple Cloning Site of pPICZa BEEN 6 Multiple Cloning Site of pl dot EEN 7 PichnaTranstormatioin TE 9 Expression an Pica iaa o a lH EES 13 A es chbensas deed deed sosbbensabsuiseedeblbbeuecenusenssud dEr inenten dansk 15 Appendix ege T 17 Recipes aabt 17 LOA A A Seen A A AR 19 PPICZA Vector atm diia allas taa 21 Lithium Chloride Transformation Method 23 Construction of In Vitro Multim rs eege onto bs hate 25 Technical Support eet measures kg dritten erediensten 33 Purchaser Notification sosiaa a R gedierte calcu Aaa 34 NOS 35 iii iv Important Information Contents Shipping Storage Reference Sources Recommended Pichia Host Strain 6 ug of each of pPICZa A B and C vector in TE buffer pH 8 0 40 ul at 150 ng ul TE buffer pH 8 0 10 mM Tris HCl 1 mM EDTA pH 8 0 The vectors are shipped o
24. Zeocin resistance Sh ble gene to allow selection of the plasmid using Zeocin To propagate and maintain the pPICZa plasmids we recommend using the following procedure 1 Use the vector in the 0 5 ug ul stock solution supplied with the kit to transform a recA endA E coli strain like TOP10 DH5a JM109 or equivalent 2 Select transformants on Low Salt LB plates containing 25 ug ml Zeocin see page 17 for a recipe 3 Prepare a glycerol stock from each transformant containing plasmid for long term storage see page 8 Continued on next page Cloning into pPICZa A B and C continued General Considerations Cloning Considerations Signal Sequence Processing Optimizing Signal Cleavage The following are some general points to consider when using pPICZa to express your gene of interest in Pichia e The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae e Many Saccharomyces genes have proven to be functional in Pichia e The premature termination of transcripts because of AT rich regions has been observed in Pichia and other eukaryotic systems Henikoff and Cohen 1984 Irniger et al 1991 Scorer et al 1993 Zaret and Sherman 1984 If you have problems expressing your gene check for premature termination by northern analysis and check your sequence for AT rich regions It may be necessary to change the sequence in order to express your gene Scorer et al 1993 e The
25. anty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 33 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 74 Pichia Pastoris Expression System 34 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany The Pichia Expression System is based on the yeast Pichia pastoris Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology Industry Associates SIBIA and Phillips Petroleum for high level expression of recombinant proteins All patents for Pichia pastoris and licenses for its use as an expression system are owned by Research Corporation Technologies RCT
26. cate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warr
27. der to ensure ligase activity Perform the reaction at 37 C T4 ligase will retain most of its activity in the restriction buffer As head to head and tail to tail multimers form they will be digested increasing the likelihood of obtaining head to tail multimers over time Ligating Multimers You are now ready to ligate the mixture of multimers generated in Step 10 into Linearized above into dephosphorylated linearized vector Vector 1 Set up the following ligation reactions Dephosphorylated vector page 28 Step 10 4 ul Expression cassette multimers Step 10 above 4 ul 10X Ligation Buffer 1 ul T4 DNA Ligase 2 5 units ul 1 ul Total volume 10 ul For the vector only control Dephosphorylated vector 4 ul Sterile water 4 ul 10X Ligation Buffer 1 ul T4 DNA Ligase 2 5 units ul 1 ul Total volume 10 ul 2 Incubate overnight at 16 C 3 You may store the ligation reactions at 20 C until ready to use or transform 1 10 pl of each ligation mix into competent E coli Note that the amount of the ligation mixture you transform depends on whether you use electrocompetent or chemically competent cells You may have to decrease the amount you to transform into electrocompetent cells to prevent arcing Continued on next page 30 Construction of n Vitro Multimers continued Transformation Remember to include the vector only and cells only controls to evaluate your into E coli experiment The vector only will indicat
28. e drug TM Store tissue culture medium containing Zeocin at 4 C in the dark Medium containing Zeocin is stable for 1 2 months pPICZa Vector Map of pPICZa The figure below summarizes the features of the pPICZa A B and C vectors The complete sequences of pPICZa A B and C are available for downloading at www invitrogen com or from Technical Support see page 33 See the next page for a description of the features of the vector Comments for pPICZa A Bg Il 3593 nucleotides Pst is in Version B only 5 AOX7 promoter region bases 1 941 e 5 AOX1 priming site bases 855 875 Ciailis in Version only a factor signal sequence bases 941 1207 Multiple cloning site bases 1208 1276 c myc epitope bases 1275 1304 Polyhistidine 6xHis tag bases 1320 1337 The two Xho sites in the vector allow 3 AOX1 priming site bases 1423 1443 the user to clone their gene in frame with AOX1 transcription termination region bases 1341 1682 the Kex2 cleavage site resulting in TEF1 promoter bases 1683 2093 expression of their native gene without EM7 promoter bases 2095 2162 additional amino acids at the N terminus Sh ble ORF bases 2163 2537 CYC1 transcription termination region bases 2538 2855 pUC origin bases 2866 3539 complementary strand Continued on next page 21 pPICZa Vector continued Features of pPICZa A B and C pPICZa A 3593 bp pPICZa B 3597 bp and pPICZa C 3598 bp contain the follow
29. e whether your vector was dephosphorylated Since the CIAP reaction is not 100 and because you often get degradation of the ends there might be a few colonies on this plate The cells only plate should have no colonies at all 1 Transform competent E coli by your method of choice using the ligation mixture from the previous page 2 After adding medium to the transformed cells and allowing them to recover plate 10 ul and 100 ul of each transformation mix onto Low Salt LB plates containing 25 ug ml Zeocin Save the remainder of your transformation mix at 4 C 3 Incubate overnight at 37 C If you do not get transformants or very few transformants plate out the remainder of the transformation mix onto Low Salt LB Zeocin plates Analyzing 1 Pick 20 transformants and inoculate each colony into 2 ml Low Salt LB Transformants containing 25 pg ml Zeocin Grow overnight at 37 C 2 Isolate plasmid DNA and digest with BglII and BamHI to release any multimers from pPICZa Be sure to include BglIJ BamHI digested pPICZa as a control It is possible to get vector rearrangements and deletions with large recombinant vectors in E coli Including Bglll BamHI digested pPICZa will allow you to detect these rearrangements deletions in the vector backbone 3 Analyze your digests on a 1 agarose gel You should see bands corresponding to 1 copy 2 copies 3 copies etc of your expression cassette along with the vector backbone
30. eactions C664 55 electrocompetent cells Continued on next page vii Accessory Products continued Plasmid Invitrogen offers a number of plasmid DNA purifications systems For more Preparation information refer to www invitrogen com or contact Technical Support page 33 Item Amount Cat no PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 50 preps K2100 05 S N A P Miniprep Kit 100 reactions K1900 01 S N A P Midiprep Kit 2 reactions K1910 01 S N A P Gel Purification Kit 25 reactions K1999 25 Detecting Fusion A number of antibodies are available from Invitrogen to detect expression of Protein your fusion protein from the pPICZa vector Horseradish peroxidase HRP conjugated antibodies allow one step detection in western blots using colorimetric or chemiluminescent detection methods The amount of antibody supplied is sufficient for 25 Westerns Antibody Epitope Cat no Anti myc Detects the 10 amino acid epitope R950 25 Anti myc HRP derived from c myc Evans et al 1985 R951 25 EQKLISEEDL Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP 6xHis tag requires the free carboxyl R931 25 group for detection Lindner et al 1997 HHHHHH COOH Purifying Fusion The polyhistidine 6xHis tag allows purification
31. er Cat no N710 02 are available separately from Invitrogen Refer to the diagrams on pages 5 7 for the sequences and location of the priming sites Once you have identified the correct clone be sure to purify the colony and make a glycerol stock for long term storage It is also a good idea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony out on a Low Salt LB plate containing 25 pg ml Zeocin Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 ml of Low Salt LB containing 25 ug ml Zeocin 3 Grow the culture to mid log phase ODs 0 5 0 7 Mix 0 85 ml of culture with 0 15 ml of sterile glycerol and transfer to a cryovial Store at 80 C Once you have cloned and sequenced your insert generate enough plasmid DNA to transform Pichia 5 10 ug of each plasmid per transformation We recommend isolating plasmid DNA using the S N A P Mini or Midiprep Kits PureLink HiPure Plasmid Mini or Midiprep Kits or CsCl gradient centrifugation see page viii for ordering information Once you have purified plasmid DNA proceed to Pichia Transformation next page Pichia Transformation Introduction Zeocin Selection Method of Transformation Pichia EasyComp Transformation Kit Important Note You should now have your gene cloned into one of the pPICZa vectors Your construct should be correctly fused to the a factor signal sequence and the C t
32. erminal peptide This section provides general guidelines to prepare plasmid DNA transform your Pichia strain and select for Zeocin resistant clones TM We generally use 100 ug ml Zeocin to select for transformants when using the X 33 Pichia strain If you are transforming your pPICZa construct into another Pichia strain note that selection conditions may vary We recommend performing a dose response curve to determine the appropriate concentration of Zeocin to use for selection of transformants in your strain We do not recommend spheroplasting for transformation of Pichia with plasmids containing the Zeocin resistance marker Spheroplasting involves removal of the cell wall to allow DNA to enter the cell Cells must first regenerate the cell wall before they are able to express the Zeocin resistance gene For this reason plating spheroplasts directly onto selective medium containing Zeocin does not yield any transformants We recommend electroporation for transformation of Pichia with pPICZa A B or C Electroporation yields 10 to 10 transformants per ug of linearized DNA and does not destroy the cell wall of Pichia If you do not have access to an electroporation device use the LiCl protocol on page 23 or the Pichia EasyComp Transformation Kit available from Invitrogen see below If you wish to perform chemical transformation of your Pichia strain with pPICZa A B or C the Pichia EasyComp Transformation Kit
33. ession Kit or Vector to a third party following written notification of and written approval from Life Technologies so that the recipient can be licensed You may not assign sub license rent lease or otherwise transfer this license agreement or any of the rights or obligation there under except as expressly permitted by Life Technologies and RCT This license agreement is effective until terminated You may terminate it at any time by destroying all Pichia Expression products in your control It will also terminate automatically if you fail to comply with the terms and conditions of the license agreement You shall upon termination of the license agreement destroy all Pichia Expression products in your control and so notify Life Technologies in writing You may contact Research Corporation Technologies at the following address Bennett Cohen Ph D Research Corporation Technologies 101 North Wilmot Road Suite 600 Tucson Arizona 85711 3335 Tel 520 748 4443 Fax 520 748 0025 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Baron M Reynes J P Stassi D and Tiraby G 1992 A Selectable Bifunctional b Galactosidase Phleomycin resistance Fusion Protein as a Potential Marker for Eukaryotic Cells Gene 114 239 243 Brake A J Merryweather J P
34. for 30 minutes without shaking Heat shock in a water bath at 42 C for 20 25 minutes Centrifuge the cells at 6000 to 8000 rpm to pellet Resuspend the pellet in 1 ml of YPD and incubate at 30 C with shaking After 1 hour and 4 hours plate 25 to 100 ul on YPD plates containing the TM appropriate concentration of Zeocin Incubate the plates for 2 3 days at 30 C Construction of n Vitro Multimers Experimental At this point you should have your gene cloned into the multiple cloning site of Outline either pPICZa A B or C To generate multiple copies of your expression cassette Stage Description 1 Digest pPICZa containing your gene of interest with BglII and BamHI to release the expression cassette Paox plus your gene 2 To clone multiple copies of the expression cassette linearize pPICZa containing your gene of interest using BamHI Note that the BamHI linearized vector already contains one copy of your expression cassette 3 Treat the BglII BamHI expression cassette with ligase in vitro Note that BglII and BamHI share 4 bases in common between their recognition sites GATC 4 Generate head to tail head to head and tail to tail multimers Head to tail ligation which is the correct orientation for expression will destroy both the BamHI and BglII sites 5 Treat the ligation mix with BamHI and BglII to eliminate head to head and tail to tail multimers
35. hat drives expression of the Zeocin resistance gene in Pichia EM7 promoter Synthetic prokaryotic promoter that drives constitutive expression of the Zeocin resistance gene in E coli Zeocin resistance gene Sh ble Allows selection of transformants in E coli and Pichia CYC1 transcription termination region GenBank accession no M34014 3 end of the Saccharomyces cerevisiae CYC1 gene that allows efficient 3 mRNA processing of the TM Zeocin resistance gene for increased stability pUC origin Allows replication and maintenance of the plasmid in E coli 22 Lithium Chloride Transformation Method Introduction Preparing Solutions Preparing Cells This is a modified version of the procedure described for S cerevisiae Gietz and Schiestl 1996 and is provided as an alternative to transformation by electroporation Transformation efficiency is between 10 to 10 cfu ug linearized DNA Lithium acetate does not work with Pichia pastoris Use only lithium chloride 1 M LiCl in distilled deionized water Filter sterilize Dilute as needed with sterile water 50 polyethylene glycol PEG 3350 in distilled deionized water Filter sterilize Store in a tightly capped bottle 2 mg ml denatured sheared salmon sperm DNA in TE Buffer pH 8 0 10 mM Tris HCl pH 8 0 1 0 mM EDTA Store at 20 C 1 Grow a 50 ml culture of Pichia pastoris in YPD at 30 C
36. igating each expression cassette stepwise see page 29 Recombinant vector rearranges and deletions are detected Construct is unstable in E coli Decrease the number of cassettes in the vector No Zeocin resistant Pichia Integration efficiency Transform using more DNA and or transformants is low do multiple transformations with more DNA and cells For More There are a number references in the literature you can consult in order to Information optimize synthesis of in vitro multimers A partial list is provided below Cohen B and Carmichael G G 1986 A Method for Constructing Multiple Tandem Repeats of Specific DNA Fragments DNA 5 339 343 Eisenberg S Francesconi S C Civalier C and Walker S S 1990 Purification of DNA Binding Proteins by Site specific DNA Affinity Chromatography Methods Enzymol 182 521 529 Graham G J and Maio J J 1992 A Rapid and Reliable Method to Create Tandem Arrays of Short DNA Sequences BioTechniques 13 780 789 Rudert W A and Trucco M 1990 DNA Polymers of Protein Binding Sequences Generated by Polymerase Chain Reaction Nucleic Acids Res 18 6460 Simpson R T Thoma F and Brubaker J M 1985 Chromatin Reconstituted from Tandemly repeated Cloned DNA Fragments and Core Histones A Model System for the Study of Higher order Structure Cell 42 799 808 Takeshita S Tezuka K i Takahashi M Honkawa H Matsuo A Matsuishi
37. ing elements All features have been functionally tested Feature Benefit 5 AOX1 promoter A 942 bp fragment containing the AOX1 promoter that allows methanol inducible high level expression of the gene of interest in Pichia Targets plasmid integration to the 40X1 locus cerevisiae a factor secretion signal from Saccharomyces Allows for efficient secretion of most proteins from Pichia Multiple cloning site Allows insertion of your gene into the expression vector c myc epitope Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Permits detection of your recombinant fusion protein with the Anti myc Antibody or Anti myc HRP Antibody Evans et al 1985 See page viii for ordering information C terminal polyhistidine 6xHis tag Permits purification of your recombinant fusion protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody Lindner et al 1997 and the Anti His C term HRP Antibody See page viii for ordering information AOX1 transcription termination TT region Native transcription termination and polyadenylation signal from AOX1 gene 260 bp that permits efficient 3 mRNA processing including polyadenylation for increased mRNA stability TEF1 promoter GenBank accession nos D12478 D01130 Transcription elongation factor 1 gene promoter from Saccharomyces cerevisiae t
38. instructions on how to prepare and use ProBond resin refer to the ProBond Purification manual TM We recommend that you use the ProBond Purification System to purify fusion proteins expressed from pPICZa A B or C see page viii for ordering information Note that instructions for equilibration of and chromatography on ProBond resin are contained in the ProBond Purification System kit If you are using a metal chelating resin other than ProBond follow the manufacturer s recommendations to purify fusion proteins expressed in bacteria or yeast TM One milliliter of ProBond resin binds at least 1 mg of recombinant protein This amount can vary depending on the protein Express your protein using a small scale culture 10 20 ml for Mut strains 100 200 ml for Mut and the optimal conditions for expression if determined Details may be found in the Pichia Expression Kit manual Once your protein is expressed separate the cells from the medium by centrifugation Store the medium at 80 C or proceed directly to purification If desired the cells can be stored at 80 C for future analysis Throughout the following protocol be sure to keep the medium and fractions on ice Small scale purifications using the 2 ml ProBond columns and buffers can be done at room temperature on the bench top For large scale purifications all reagents must be kept at 4 C Continued on next page 15 Purification continued
39. is Gene 136 111 119 Tschopp J F Brust P F Cregg J M Stillman C and Gingeras T R 1987a Expression of the lacZ Gene from Two Methanol Regulated Promoters in Pichia pastoris Nucleic Acids Res 15 3859 3876 Zaret K S and Sherman F 1984 Mutationally Altered 3 Ends of Yeast CYC1 mRNA Affect Transcript Stability and Translational Efficiency J Mol Biol 177 107 136 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 35 Notes 36 Notes 37 Notes 38 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
40. is available from Invitrogen see vii for ordering information The Pichia EasyComp Transformation Kit provides reagents to prepare 6 preparations of competent cells Each preparation will yield enough competent cells for 20 transformations Competent cells may be used immediately or frozen and stored for future use For more information refer to www invitrogen com or contact Technical Support page 33 Since pPICZa does not contain the HIS4 gene integration can only occur at the AOX1 locus Vector linearized within the 5 AOX1 region will integrate by gene insertion into the host 5 AOX1 region Therefore the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol Mut or not Mut To generate a Mut recombinant strain you must use a Pichia host that contains the native AOX1 gene e g X 33 SMD1168H If you wish to generate a Mut recombinant strain then use a Pichia host that has a disrupted AOX1 gene i e KM71H The pPICZa vectors do not contain a yeast origin of replication Transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome Continued on next page Pichia Transformation continued Before Starting Linearizing Your pPICZa Construct Restriction Digest 10 You will need the following reagents for transforming Pichia and selecting transformants on Zeocin Note Inclusion of sorbitol in YPD plates stabilizes electr
41. n method alkaline phosphatase horseradish peroxidase radiolabeled antibody Anti myc antibodies see the next page Anti His C term antibodies see the next page Functional assay Varies depending on assay Varies depending on assay Used to compare relative amounts of protein Continued on next page 13 Expression in Pichia continued Polyacrylamide Gel Electrophoresis Western Analysis Important Expression Guidelines 14 To facilitate separation and visualization of your recombinant protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels are available from Invitrogen In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits For more information about the appropriate gels standards and stains to use to visualize your recombinant protein refer to our website at www invitrogen com or call Technical Support page 33 To detect expression of your recombinant fusion protein by western blot analysis you may use the Anti myc antibodies or the Anti His C term antibodies available from Invitrogen see page viii for ordering information or an antibody to your protein of interest In addition the Positope Control Protein Cat no R900 50 is available from Invitrogen for use as a positive control for detection of fusion proteins containing a c myc epitope or a polyhi
42. n 900 ml of water Add 20 g of agar Autoclave for 20 minutes on liquid cycle Add 100 ml of 20 dextrose filter sterilize dextrose before use TM Cool solution to 60 C and add the appropriate amount of Zeocin from a 100 mg ml stock solution A A Note It is necessary to include Zeocin in the medium for selection of Pichia transformants only Zeocin may be omitted from the medium when performing expression studies Store YPDS slants or plates containing Zeocin at 4 C The shelf life is one to two weeks Zeochn Zeocin Molecular Weight Formula and Structure Applications of Zeocin Zeocin is a member of the bleomycin phleomycin family of antibiotics isolated from Streptomyces Antibiotics in this family are broad spectrum antibiotics that act as strong anti bacterial and anti tumor drugs They show strong toxicity against bacteria fungi including yeast plants and mammalian cells Baron et al 1992 Drocourt et al 1990 Mulsant et al 1988 Perez et al 1989 The Zeocin resistance protein has been isolated and characterized Calmels et al 1991 Drocourt et al 1990 This protein the product of the Sh ble gene Streptoalloteichus hindustanus bleomycin gene is a 13 7 kDa protein that binds Zeocin and inhibits its DNA strand cleavage activity Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin The formula for Zeocin is CsoHs N2102153 a
43. n wet ice and should be stored at 20 C The pPICZa A B and C vectors may be used with the Original Pichia Expression Kit Cat no K1710 01 and are included in the EasySelect Pichia Expression Kit Cat no K1740 01 available from Invitrogen Additional general information about recombinant protein expression in Pichia pastoris is provided in the manuals for the Original Pichia Expression Kit and the EasySelect Pichia Expression Kit The manuals can be downloaded from our Website www invitrogen com or obtained by calling Technical Support see page 33 For more information about the Original Pichia Expression Kit or the EasySelect Pichia Expression Kit refer to our Website or contact Technical Support More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference see page vii for ordering information Higgins D R and Cregg J M 1998 Pichia Protocols In Methods in Molecular Biology Vol 103 J M Walker ed Humana Press Totowa NJ We recommend using the X 33 Pichia strain as the host for expression of recombinant proteins from pPICZa Other Pichia strains are suitable The X 33 Pichia strain is available from Invitrogen see page vii for ordering information and has the following genotype and phenotype Genotype Wild type Phenotype Mut Continued on next page Important Information continued Materials Needed vi For the procedures de
44. nd the molecular weight is 1 535 The diagram below shows the structure of Zeocin CH3 H NH N R HN AN be 2 NH MW 1 535 Zeocin is used for selection in mammalian cells Mulsant et al 1988 plants Perez et al 1989 yeast Baron et al 1992 and prokaryotes Drocourt et al 1990 Suggested concentrations of Zeocin for selection in Pichia and E coli are listed below TM Organism Zeocin Concentration and Selective Medium E coli 2550 ug ml in Low Salt LB medium see page 17 for a recipe Pichia 100 1000 pg ml varies with strain and medium Efficient selection requires that the concentration of NaCl be no more than 5 g L lt 90 mM Continued on next page 19 Zeocin continued Handling Zeocin 20 TM High salt and acidity or basicity inactivate Zeocin therefore we recommend that you reduce the salt in bacterial medium and adjust the pH to 7 5 to keep the drug active see Low Salt LB Medium page 17 Note that the salt concentration and pH do not need to be adjusted when preparing tissue culture medium containing Zeocin TM Store Zeocin at 20 C and thaw on ice before use Zeocin is light sensitive Store drug plates and medium containing drug in the dark Wear gloves a laboratory coat and safety glasses or goggles when handling solutions containing Zeocin Zeocin is toxic Do not ingest or inhale solutions containing th
45. nsformation Method Maintenance of Plasmids The pPICZa vector is supplied with the multiple cloning site in three reading frames A B and C to facilitate cloning your gene of interest in frame with the C terminal peptide containing the c myc epitope and a polyhistidine 6xHis tag Use the diagrams provided on pages 5 7 to help you design a strategy to clone your gene of interest in frame with the a factor secretion signal and the C terminal peptide General considerations for cloning and transformation are discussed in this section For assistance with E coli transformations restriction enzyme analysis DNA biochemistry and plasmid preparation refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Many E coli strains are suitable for the propagation of the pPICZa vectors including TOP10 Cat no C610 00 JM109 and Do We recommend that you propagate the pPICZa vectors in E coli strains that are recombination deficient recA and endonuclease A deficient endA For your convenience TOP10 E coli are available as chemically competent or electrocompetent cells from Invitrogen See page vii for ordering information You may use any method of choice for transformation Chemical transformation is the most convenient for many researchers Electroporation is the most efficient and the method of choice for large plasmids The pPICZa vectors contain the
46. oducts listed in this section are intended for use with the pPICZa vectors For more information refer to www invitrogen com or call Technical Support see page 33 Obtaining Zeocin Zeocin may be obtained from Invitrogen For your convenience the drug is prepared in autoclaved deionized water and available in 1 25 ml aliquots at a concentration of 100 mg ml The stability of Zeocin is guaranteed for six months if stored at 20 C Amount Catalog no lg R250 01 58 R250 05 Accessory Many reagents that may be used with the pPICZa vectors and for Pichia Products expression are available from Invitrogen Ordering information is provided below Item Amount Cat no X 33 Pichia strain 1 stab C180 00 KM71H Pichia strain 1 stab C182 00 SMD1168H Pichia strain 1 stab C184 00 DI AOX1 Pichia Primer 2 ug N710 02 3 AOX1 Pichia Primer 2ug N720 02 pPICZ A B and C 20 ug each V190 20 pPIC6a A B and C 20 ug each V215 20 pPIC6a Starter Kit 1 kit K215 01 pPIC6 A B and C 20 ug each V210 20 pPIC6 Starter Kit 1 kit K210 01 Original Pichia Expression Kit 1 kit K1710 01 EasySelect Pichia Expression Kit 1 kit K1740 01 Pichia EasyComp Transformation Kit 1 kit K1730 01 Pichia Protocols 1 book G100 01 One Shot TOP10 20 reactions C4040 03 chemically competent cells One Shot TOP10 Electrocomp 20 reactions 4040 52 electrocompetent cells Electrocomp TOP10 20 r
47. on Precipitate the DNA by adding 5 ul of 3 M sodium acetate and 110 ul of 100 ethanol Incubate on ice for 30 minutes Centrifuge at maximum speed in a microcentrifuge for 10 minutes at 4 C Carefully decant the supernatant Wash the nucleic acid pellet with 80 ethanol centrifuge 2 minutes and remove the ethanol Centrifuge again for 1 minute remove residual ethanol and air dry the pellet Resuspend pellet in 8 pl sterile water Save on ice if you plan to ligate your insert immediately see Ligation and Digestion of Expression Cassette next page or store at 20 C Continued on next page Construction of n Vitro Multimers continued Ligating and Ligation of the expression cassette will generate head to tail head to head and Digesting tail to tail multimers Creation of head to tail multimers will be in the correct Expression orientation for expression and will destroy both the BamHI and BglII sites Cassette between the expression cassettes Digestion of the multimers with BamHI and BgllI will eliminate those multimers with tail to tail and head to head orientation After digestion with these two restriction enzymes you will have a mixture of multimers containing 1 2 3 etc copies of your gene that can be ligated into BamHI linearized recombinant pPICZa 1 Set up a 20 ul ligation reactions as follows Bglll BamHI digested expression cassette 15 pl Sterile water 2 ul 10X Ligation Buffer with ATP 2 ul T4 DNA Ligase
48. ontinue incubating the other culture for three more hours for a total of four hours and then plate out all of the cells by spreading 200 ul on TM 150 mm plates containing the appropriate concentration of Zeocin 6 Incubate plates for 2 to 4 days at 30 C until colonies form If you used a Pichia strain containing a native AOX1 gene e g X 33 GS115 SMD1168H as the host for your pPICZa construct your Zeocin resistant transformants will be Mut If you used a strain containing a deletion in the AOX1 gene e g KM71H your transformants will be Mut If you wish to verify the Mut phenotype of your Zeocin resistant transformants you may refer to the general guidelines provided in the EasySelect Pichia Expression Kit manual or the Original Pichia Expression Kit manual or to published reference sources Higgins and Cregg 1998 You are now ready to test your transformants for expression of your gene of interest See Expression in Pichia next page Expression in Pichia Introduction Important Detecting Recombinant Proteins in Pichia The primary purpose of small scale expression is to identify confirm a recombinant Pichia clone that is expressing the correct protein Small scale expression conditions may not be optimal for your protein For this reason the method you choose for detection e g SDS PAGE western or functional assay may be an important factor in determining the success of expression If your method
49. oporated cells as they appear to be somewhat osmotically sensitive e 5 10 ug pure pPICZa containing your insert e YPD Medium e 50 ml conical polypropylene tubes e 1 liter cold 4 C sterile water place on ice the day of the experiment e 25 ml cold 4 C sterile 1 M sorbitol place on ice the day of the experiment e 30 C incubator e Electroporation device and 0 2 cm cuvettes e YPDS plates containing the appropriate concentration of Zeocin see page 18 for recipe To promote integration we recommend that you linearize your pPICZa construct within the 5 AOX1 region The table below lists unique sites that may be used to linearize pPICZa prior to transformation Other restriction sites are possible Note that for the enzymes listed below the cleavage site is the same for versions A B and C of pPICZa Be sure that your insert does not contain the restriction site you wish to use to linearize your vector Enzyme Restriction Site bp Supplier Sac I 209 Many Pme I 414 New England Biolabs BstX I 707 Many 1 Digest 5 10 ug of plasmid DNA with one of the enzymes listed above 2 Check a small aliquot of your digest by agarose gel electrophoresis for complete linearization 3 If the vector is completely linearized heat inactivate or add EDTA to stop the reaction phenol chloroform extract once and ethanol precipitate using 1 10 volume 3 M sodium acetate and 2 5 volumes of 100 ethanol Centrifuge
50. pecific Pichia proteins Be sure to save all fractions washes and flow through for analysis by SDS PAGE You may need to use western blot analysis to detect your protein if expression is low or not enough protein was loaded onto the column Refer to the ProBond Purification System manual for a guide to troubleshoot chromatography You may find it necessary to scale up your purification to obtain sufficient amounts of purified protein Adjust the pH and NaCl concentration of your lysate with 1 10 volume of 10X Stock Solution B ProBond Purification System before adding it to the column The pH should be gt 7 5 and the NaCl concentration should be 500 mM Using 10X Stock Solution B to adjust the pH and the ionic strength keeps the total volume small for sample application Recipes Pre mixed Expression Media Low Salt LB Medium with Zeocin YPD Zeocin Appendix The table below lists the pre mixed media and media components available from Invitrogen specifically for Pichia Please contact Technical Support see page 33 for more information Item Amount Cat no Yeast Nitrogen Base 67 g pouch Q300 07 with ammonium sulfate Each pouch contains reagents to without amino acids prepare 500 ml of a 10X YNB solution 500 g Q300 09 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 ml Adjust pH to 7 5 with IN NaOH
51. port see page 33 For a map and a description of the features of pPICZa refer to pages 21 22 5 end of AOX7 mRNA 5 AOX7 priming site AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TIGCGACTGG TTCCAATTGA BESTEHEN CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT I ATTCGAAACG ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA GCA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala a factor signal sequence CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG TTT Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Xho L T ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA TCT CTC Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Kex2 signal cleavage PstI EcoRI Pmil Sfi l BsmB fi I GAG AAA AGA GAG GCT GAA GC TGCAG GAATTCAC GTGGCCCAG CCGGCCGTC TCGGA Glu Lys Arg Glu Alajciu Ala Ste13 signal cleavage Asp718 Kpn Xho Sac ll Not Xba c myc epitope l lo l l l TCGGTACCTC GAGCCGCGGC GGCCGCCAGC TTTCTA GAA CAA AAA CTC ATC TCA GAA Glu Gln Lys Leu Ile Ser Glu polyhistidine tag GAG GAT CTG AAT AGC GCC GTC GAC
52. rg may be more efficient when followed by Glu Ala repeats A number of amino acids are tolerated at site X instead of Glu in the sequence Glu Lys Arg X These amino acids include the aromatic amino acids small amino acids and histidine Proline however will inhibit Kex2 cleavage For more information on Kex2 cleavage see Brake et al 1984 There are some cases where Ste13 cleavage of Glu Ala repeats is not efficient and Glu Ala repeats are left on the N terminus of the expressed protein of interest This is generally dependent on the protein of interest Continued on next page Cloning into pPICZa A B and C continued Expressing If you wish to have your protein expressed with a native N terminus you Recombinant should clone your gene flush with the Kex2 cleavage site Use PCR to rebuild Protein with a the sequence from the Xho I site at bp 1184 1189 to the arginine codon at Native N terminus nucleotides 1193 1195 Remember to include the first amino acid s of your protein if necessary for correct fusion to the Kex2 cleavage site Constructing pPICZa A B and C contain unique BglII and BamHI sites to allow construction Multimeric of plasmids containing multiple copies of your gene For information on how to Plasmids construct multimers refer to pages 25 32 Continued on next page Cloning into pPICZa A B and C continued Multiple Cloning Below is the multiple cloning site for pPICZa A Restriction sites are labeled to
53. scribed in this manual you will need the following reagents and equipment Additional reagents may be required Please check each experiment to ensure you have all the reagents necessary See pages vii viii for ordering information Equipment e Microbiological equipment e Electroporation device and 0 2 cm cuvettes or reagents for transformation e 16 C 37 C and 65 C water baths or temperature blocks e 30 C and 37 C shaking and non shaking incubators e Hemacytometer e Microtiter plates optional Reagents e Pichia host strain e g X 33 SMD1168H KM71H e Electrocompetent or chemically competent E coli must be recA endA for transformation e Restriction enzymes and appropriate buffers e Agarose and low melt agarose e SNAP Gel Purification Kit or glass milk e Sterile water e CIAP calf intestinal alkaline phosphatase 1 unit ul e 10X CIAP Buffer e Phenol chloroform e 3 M sodium acetate e 100 ethanol e 80 ethanol e T4 Ligase 2 5 units pl e 10X Ligation Buffer with ATP e Low Salt LB medium see page 17 for recipe e Zeocin selection agent see page vii for ordering information e Low Salt LB plates containing 25 ug ml Zeocin see page 17 for recipe e YPDS plates containing the appropriate concentration of Zeocin see page 18 for recipe e 50 ml conical centrifuge tubes e 15 ml polypropylene tubes e Optional ProBond Purification System Accessory Products Introduction The pr
54. stidine 6xHis tag WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods For more information refer to our website at www invitrogen com or call Technical Support page 33 Because the pPICZa vector does not contain the HIS4 gene his4 Pichia strains containing the integrated plasmid must be grown in medium containing 0 004 histidine If histidine is not present in the medium the cells will not grow If you use X 33 SMD1168H or KM71H as the host strain supplementation of the medium with histidine is not required General guidelines to perform small scale expression optimize expression and scale up of expression are provided in the EasySelect Pichia Expression Kit manual or the Original Pichia Expression Kit manual Purification Introduction ProBond Resin Binding Capacity of ProBond Expressing Secreted Protein Important In this section you will grow and induce a 10 200 ml culture of your Pichia transformant for trial purification on a metal chelating resin such as ProBond You may harvest the cells and store both the supernatant medium and the cells at 80 C until you are ready to purify your fusion protein or you may proceed directly with protein purification Note that this section only describes preparation of cell lysates and sample application onto ProBond For
55. ur protein with a native N terminus you must use PCR and utilize the Xho I site upstream of the Kex2 cleavage site to clone your gene flush with the Kex2 cleavage site see page 4 for more details Continued on next page 7 Cloning into pPICZa A B and C continued E coli Transformation Important N N EVO 7 O O QE NOT Preparing a Glycerol Stock Plasmid Preparation Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 DH54 JM109 and select on Low Salt LB agar plates containing 25 pg ml Zeocin see below Note that there is no blue white screening for the presence of insert with pPICZa A B or C Once you have obtained Zeocin resistant colonies pick 10 transformants and screen for the presence and orientation of your insert To facilitate selection of Zeocin resistant E coli the salt concentration of the medium must remain low lt 90 mM and the pH must be 7 5 Prepare Low Salt LB broth and plates using the recipe in the Appendix page 17 Failure to lower the salt content of your LB medium will result in non selection due to inhibition of the drug We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression and cloned in frame with the a factor signal sequence and the C terminal peptide To facilitate sequencing the 3 AOX1 Pichia Primer Cat no N720 02 and the 5 AOX1 Pichia Prim
56. urification Kit see page vii or glass milk e Sterile water e CIAP calf intestinal alkaline phosphatase 1 unit ul Boehringer Mannheim e 10X CIAP Buffer e Phenol chloroform e 3M sodium acetate e 100 ethanol e 80 ethanol e T4 Ligase 2 5 units ul e 10X Ligation Buffer with ATP e Low Salt LB plates containing 25 ug ml Zeocin e 150 mm plates for plating transformants e 16 C 37 C and 65 C water baths or temperature blocks Controls In order to evaluate your transformants and expression data later on we recommend transforming Pichia with pPICZa the parent vector and pPICZa containing one copy of your gene of interest This will allow you to compare expression levels to see if multiple copies significantly increase the amount of protein produced Also if you elect to determine how many copies of your gene are in a recombinant by dot or Southern blot the strain with the parent vector will control for background hybridization and the strain with the single copy gene will provide a signal to normalize your data Continued on next page 26 Construction of n Vitro Multimers continued Once you have created a pPICZa plasmid containing multimers Note that this Important plasmid cannot be linearized because any enzyme that cuts in the 5 AOX1 region will cut in all of the 5 AOX1 regions present in the multimer You can transform with uncut plasmid but you will need to use 50 100 ug of DNA to compensate for the 1
57. xpression Products or the Expression Kit to facilitate or advance research or development directed to a Commercial Product and any use of Expression Products or the Expression Kit to facilitate or advance any research or development program the results of which will be directly applied to the development or manufacture of a Commercial Product Expression Products means products expressed with the Expression Kit or with the use of any Pichia expression vectors including the Expression Vector or host strains Commercial Product means any product intended for sale or commercial use Commercial entities may conduct their evaluation for one year at which time this license automatically terminates Commercial entities will be contacted by Research Corporation Technologies during the evaluation period regarding their desire for a commercial license Access to the Expression Kit and Vector must be limited solely to those officers employees and students of your institution who need access to perform the above described research or evaluation You must inform each such officer employee and student of the provisions of this license agreement and require them to agree in writing to be bound by the provisions of this license agreement You may not distribute any Expression Vector or host strain contained herein or in the Expression Kit to others even those within your own institution You may only transfer modified altered or original material from the Expr

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