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pPICZ A, B, and C - Thermo Fisher Scientific

1. bne el CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT Sfu EcoR Pml Sfi BsmB Asp718 Kpn Xho I I I I I I I I ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC Sac Il Not SnaB myc epitope I I d GGCGGCCGCC AGCTT JACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Polyhistidine tag l l AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA Asn Ser Ala Val Asp His His His His His His CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA 3 AOX1 priming site GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT 3 polyadenylation site TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC Continued on next page Cloning into pPICZ A B and C Continued E coli Transformation Important Preparing a Glycerol Stock Plasmid Preparation Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 DH5 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 pPICZ 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
2. 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 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 with shaking to an ODe of 0 8 to 1 0 approximately 10 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 6 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 1 Boil a 1 ml sample of single stranded DNA for 5 minutes then quickly chill on ice Keep on ice Note It is not necessary nor desirable to boil the carrier DNA prior to each use Store a small aliquot at 20 C and boil every 3 4 times the DNA is thawed 2 Centrifuge the cells from Step 6 above and remove the LiCl with a pipet 3 Foreach transformation add the following reagents in
3. 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 TM 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 the drug Store tissue culture medium containing Zeocin at 4 C in the dark Medium TM containing Zeocin is stable for 1 2 months Map and Features of pPICZ A B and C Map of pPICZ The figure below summarizes the features of the pPICZ A B and C vectors The A B and C complete sequences for pPICZ A B and C are available for downloading from our web site at www invitrogen com or from Technical Support page 32 See the next page for a description of the features of the vector c myc epitope 6xHis Stop Comments for pPICZ A 3329 nucleotides 5 AOX1 promoter region bases 1 941 5 end of AOX1 mRNA base 824 The restriction site 5 AOX1 priming site bases 855 875 between Not and the Multiple cloning site bases 932 1011 myc epitope is different c myc epitope tag bases 1012 1044 in each version of pPICZ Polyhis
4. A B and C Continued General Considerations Cloning Considerations Construction of Multimeric Plasmids The following are some general points to consider when using pPICZ 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 amp Cohen 1984 Irniger et al 1991 Scorer et al 1993 Zaret amp 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 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 For proper initiation of translation your insert should contain an initiation ATG codon as part of a yeast consensus sequence Romanos et al 1992 An example of a yeast consensus sequence is provided below The ATG initiation codon is shown underlined G A NNATGG To express your gene as a recombinant fusion protein you must clone your gene in frame with the C terminal p
5. 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 pPICZ A B or C the Pichia EasyComp Transformation Kit is available from Invitrogen see page v 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 visit our web site at www invitrogen com or contact Technical Support page 32 Since pPICZ 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 GS115 SMD1168H If you wish to generate a Mut recombinant strain then use a Pichia host that has a disrupted AOX1 gene i e KM71H Continued on next page Pichia Transformation Continued His4 Host Strains Note Materials Needed Linearizing Your pPICZ Constr
6. from Invitrogen page v allows you to rapidly purify DNA fragments from regular agarose gels Alternatively you may use glass milk To use the S N A P Gel Purification Kit follow the steps below 1 Electrophorese your BamH I Bel II 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 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 PureLink or S N A P spin column Centrifuge 1 minute at 3000 x g in a microcentrifuge and discard the supernatant 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 Construction of n Vitro Multimers Continued Dephosphorylation Dephosphorylation of the BamH I digested vector is necessary to prevent self ligation of Vector 1 ON Hr BS 8 10 Take your BamH I digest from Digestion of Recombinant pPICZ Step 2 and phenol extract then ethanol precipitate th
7. invitrogen com or contact Technical Support page 32 Continued on next page V Accessory Products Continued Zeocin Detection of Fusion Protein Purification of Fusion Protein vi Zeocin may be obtained from Invitrogen see above 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 A number of antibodies are available from Invitrogen to detect expression of your fusion protein from the pPICZ 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 Western Blots Antibody Epitope Catalog no Anti myc Detects the 10 amino acid epitope R950 25 Anti myc HRP derived from c myc Evans et al 1985 R951 25 EOKLISEEDL 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 The polyhistidine 6xHis tag allows purification of the recombinant fusion protein using metal chelating resins such as ProBond Ordering information for ProBond resin is provided below Product Quantity Catalog no Pro
8. low 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 C terminal peptide if desired 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 an 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 pg 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 5 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 PureLink Quick Plasmid Miniprep Kit or the PureLink HiPure Plasmid Midiprep Kit page v or CsCl gradient centrifugation Once you have purified plasmid DNA proceed to Pichia Tran
9. 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 Important Preparation of Cell Lysates 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 page vi You may harvest the cells and store them at 80 C until you are ready to purify your fusion protein or you may proceed directly with protein purification Note This section only describes preparation of cell lysates and sample application onto ProBond For instructions on how to prepare and use ProBond resin refer to the ProBond Purification System manual TM TM We recommend that you use the ProBond Purification System page vi to purify fusion proteins expressed from pPICZ A B or C The ProBond Purification kit contains six 2 ml precharged prepacked ProBond resin columns buffers for native and denaturing purification and an instruction manual Note Instructions for equilibration of and chromatography on ProBond resin are contained in the ProBond Purification Kit If you ar
10. 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 Remember to include the vector only and cells only controls to evaluate your experiment The vector only will indicate 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 Transform competent E coli by your method of choice 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 pg ml Zeocin page 17 Save the remainder of your transformation mix at 4 C 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 Continued on next page 29 Construction of n Vitro Multimers Continued Analysis of To analyze your transformants Transformants 1 Pick 20 transformants and inoculate each colony into 2 ml Low Salt LB TM containing 25 pg ml Zeocin page 17 Grow overnight at 37 C 2 Isolate plasmid DNA and digest with Bgl II and BamH I to release any multimers from pPICZ Note Be sure to incl
11. 5 10 minutes at 12 000 x g 6 Transfer the clear supernatant to a fresh container and analyze for your protein The total protein concentration should be around 2 3 mg ml 7 Save the pellet and extract with 6 M urea or 1 Triton X 100 to check for insoluble protein Continued on next page 15 Purification Continued Sample Application Native Conditions Sample Application Denaturing Conditions Note Analysis of Purification Scale up 16 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 2 3 mg ml total protein of Pichia lysate 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 ly
12. 90 20 Note that the pPICZ expression vectors are supplied in suspension Component Quantity Composition pPICZ A Expression Vector 20 ug 40 pl of 0 5 pg pl vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 pPICZ B Expression Vector 20 ug 40 pl of 0 5 pg pl vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 pPICZ C Expression Vector 20 ug 40 pl of 0 5 ug l vector in 10 mM Tris HCl 1 mM EDTA pH 8 0 GS115 pPICZ lacZ Positive 1 stab zx Control strain Shipping Storage The components included with Catalog no V190 20 are shipped on wet ice Upon receipt store as directed below For long term storage of your positive control stab strain we recommend preparing a glycerol stock immediately upon receipt and storing at 80 C Component Shipping Storage pPICZ A Expression Vector Wet ice Store at 20 C pPICZ B Expression Vector Wet ice Store at 20 C pPICZ C Expression Vector Wet ice Store at 20 C GS115 pPICZ lacZ positive control strain Wet ice Store at 4 C iv Accessory Products Additional Products E Gel Agarose Gels The products listed in this section are intended for use with the pPICZ vectors For more information visit our web site at www invitrogen com or contact Technical Support page 32 Product Quantity Catalog no X 33 Pichia strain 1 stab C180 00 GS115 Pichia strain 1 s
13. Bond Purification System 6 purifications K850 01 ProBond Purification System with Anti myc 1 Kit K852 01 HRP Antibody ProBond Purification System with Anti 1 Kit K853 01 His C term HRP Antibody ProBond Nickel Chelating Resin 50 ml R801 01 150 ml R801 15 Purification Columns 50 each R640 50 Overview Introduction Reference Sources Recommended Pichia Host Strain Introduction pPICZ A B and C are 3 3 kb expression vectors used to express recombinant proteins in Pichia pastoris Recombinant proteins are expressed as fusions to a C terminal peptide containing the c myc epitope and a polyhistidine 6xHis tag The vector allows high level methanol inducible expression of the gene of interest in Pichia and can be used in any Pichia strain including X33 GS115 SMD1168H and KM71H pPICZ 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 Zeocin resistance gene for selection in both E coli and Pichia Baron et al 1992 Drocourt et al 1990 e C terminal peptide containing the c myc epitope and a polyhistidine 6xHis tag for detection and purification of a recombinant fusion protein if desired e Three reading frames to facilitate in frame cloning with the C terminal peptide The pPICZ A B and C expression vectors may
14. EcoR Pml Sfi BsmB Asp718 1 Kpn Xho I l l l l l l l ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC Sac Il Not Xba myc epitope l l GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Polyhistidine tag l AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT Asn Ser Ala Val Asp His His His His His His CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG 3 AOX7 priming site GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT 3 polyadenylation site TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TGA GTTTGTAGCC TTAGACATGA KKK TCTTGCTAGA TTCTAATCAA CATTTTTGAT ACTTTTTTAT ETO Continued on next page Cloning into pPICZ A B and C Continued Multiple Cloning Below is the multiple cloning site for pPICZ C Restriction sites are labeled to Site of pPICZ C indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing You can download the complete sequence of pPICZ C from our web site at www invitrogen com or by contacting Technical Support see page 32 For a map and a description of the features of pPICZ refer to the Appendix pages 21 22 5 end of AOX1 mRNA 5 AOX7 priming site 811 AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA 871 931 991 1041 1097 1157 1217
15. Invitrogen by technologies pPICZ A B and C Pichia expression vectors for selection on Zeocin and purification of recombinant proteins Catalog no V190 20 Rev Date 7 July 2010 Manual part no 25 0148 MAN00000034 ii Table of Contents Kit COMPEMES AN Storage istos eec ote ies rie o eee eei indes de i sand end degen rait deer a disease iv Accessory Productserie vaten eio oibus nid ui edi edant e etii ped itd v Dent OG KOK akai a IPAE ete E E T E E E ex vo bee SE 1 OMAS Ae 1 TE lr TEE 3 Cloning into pPICZ A B and Crp a REENEN 3 TR Ee Ee e EE 9 Expression iiPielta c i staat sanete ade dta i e e dated Me anneke daat 13 Puxificallon z aiite eee eei EM E MODE eo ae dte a eb e OE 15 Appendix oen ae ate tutes neme Enna aenema UO aT Roten iesu bennen wenende isis eren annae 17 ReGipes 17 Zeocl os cnm d OU TER cene xr tte tbe RU EIS 19 Map and Features of pPICZ A B and Ces 21 Lithium Chloride Transformation Method 23 Construction of In Vitro Multimers annae eneen envenvensennenneenvenvenvenvenvenvenneenennsenvenvenvenvenveenenneenvenenneenven 24 Technical Support wasmand eneen petu ade aen an tet i 32 Purchaser Notification eec dne d tee RE e er EH EU i RE oh a Toa a aaa aeaea aa isede atiet 33 References 2 vias 5e o en e ce e tee o b E e Reate e eit ae edere eae 34 iii Kit Contents and Storage Contents The following components are included with Catalog no V1
16. ar Cloning A Laboratory Manual Second Ed Cold Spring Harbor Laboratory Press Plainview New York 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 pastoris 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 34 Notes 35 Notes 36 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
17. be used with the Original Pichia Expression Kit and are included in the EasySelect Pichia Expression Kit see page v for ordering information 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 For more information about the Original Pichia Expression Kit the EasySelect Pichia Expression Kit or their manuals visit our web site at www invitrogen com or contact Technical Support page 32 More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference 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 see page v for ordering information We recommend using the X 33 Pichia strain as the host for expression of recombinant proteins from pPICZ Other Pichia strains including GS115 KM71H and SMD1168H are suitable The X 33 Pichia strain and other strains are available from Invitrogen see page v for ordering information The X 33 Pichia strain has the following genotype and phenotype Genotype Wild type Phenotype Mut Overview Continued Experimental The following table describes the basic steps needed to clone and express your Overview gene of interest in pPICZ Step Action 1 P
18. c Il Not Apa myc epitope 991 GGCGGCCGCC AGCTT GEGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG Glu Glin Lys Leu Ile Ser Glu Glu Asp Leu Polyhistidine tag i 1042 AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTTAGCCT TAGACATGAC Asn Ser Ala Val Asp His His His His His His 1098 TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG 3 AOX1 priming site 1 1158 AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATT 3 polyadenylation site 1218 TGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT Continued on next page Cloning into pPICZ A B and C Continued Multiple Cloning Site of pPICZ B 811 871 931 991 1040 1096 1156 1216 Below is the multiple cloning site for pPICZ B Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing You can download the complete sequence of pPICZ B from our web site at www invitrogen com or by contacting Technical Support see page 32 For a map and a description of the features of pPICZ refer to the Appendix pages 21 22 5 end of AOX1 mRNA 5 AOX7 priming site AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA PRECARI CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT Sfu
19. combinant Pichia clone in medium containing 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 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 end of your protein and the C terminal tag Silver stained Technique Method of Detection Sensitivity SDS PAGE Visualization by eye Can detect as little as 100 ng in Coomassie stained a single band SDS PAGE Visualization by eye Can detect as little as 2 ng in a single band Western Analysis Antibody to your particular protein Can detect as little as 1 10 pg depending on detection 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 Tris Glycine polyacrylamide gel
20. d the pellet in 20 ml of ice cold 0 4 C 1 M sorbitol Centrifuge the cells as in Step 3 then resuspend the pellet in 1 ml of ice cold 0 4 C 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 ug of linearized pPICZ DNA in 5 10 pl sterile water and transfer them to an ice cold 0 4 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 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 TM appropriate concentration of Zeocin Incubate plates for 2 3 days at 30 C until colonies form Pick 10 20 colonies and purify streak for single colonies on fresh YPD or 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 be
21. e DNA Resuspend in 17 pl of sterile water Set up a 20 ul dephosphorylation reaction in a microcentrifuge tube as follows e 17 ul BamH I digested recombinant pPICZ page 24 e 2 ul 10X CIAP Buffer e 1 ulCIAP 1 Unit ul 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 ul of phenol chloroform and extract your DNA solution 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 ul sterile water Save on ice if you plan to ligate your insert immediately see Ligation and Digestion of Expression Cassette or store at 20 C Continued on next page 21 Construction of n Vitro Multimers Continued Ligation and Digestion of Expression Cassette Note 28 Ligation of the expression cassette will generate head to tail head to head and tail to tail multimers Creation of head to tail multimers will be in the correct orientation for expression and will destroy both the BamH I and Bgl II sites between the expression cassettes Digestion of the multimers with BamH I and Bgl II will eliminate those
22. e 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 Throughout the following protocol be sure to keep the cell lysate and fractions on ice Small scale purifications using the 2 ml ProBond columns and buffers can be performed at room temperature on the bench top For large scale purifications all reagents must be kept at 4 C 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 Refer to the Pichia Expression Kit manual for details Once your protein is expressed follow the protocol below to prepare a cell lysate for chromatography on ProBond Prepare Breaking Buffer BB as described in the Appendix page 18 1 Wash cells once in BB by resuspending them and centrifuging 5 10 minutes at 3000 x g at 4 C 2 Resuspend the cells to an OD soo of 50 100 in BB 3 Add an equal volume of acid washed glass beads 0 5 mm Estimate volume by displacement 4 Vortex the mixture for 30 seconds then incubate on ice for 30 seconds Repeat 7 more times Alternating vortexing with cooling keeps the cell extracts cold and reduces denaturation of your protein 5 Centrifuge the sample at 4 C for
23. east extract e 182 2 g sorbitol e 20g of peptone Add 20 g of 2 agar to the solution and dissolve Autoclave for 20 minutes on liquid cycle Add 100 ml of 2 dextrose filter sterilize dextrose before use Hi Fe ES S Cool solution to 60 C and add the appropriate amount of Zeocin from a 100 mg ml stock solution 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 TM 6 Store YPDS slants or plates containing Zeocin at 4 C The shelf life is one to two weeks 50 mM sodium phosphate pH 7 4 1 mM EDTA 5 glycerol Sterile water 1 mM PMSF phenylmethylsulfonyl fluoride You may use other protease inhibitors 1 Prepare a stock solution of your desired protease inhibitors and store appropriately Follow manufacturer s recommendations 2 For 1 liter dissolve the following into 900 ml water e 6g sodium phosphate monobasic e 372mgEDTA e 50 ml glycerol Use NaOH to adjust pH and bring up the volume to 1 liter Store at 4 C Add 1 mM PMSF or other protease inhibitors immediately before use 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 dru
24. ector 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 Expression 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 Tech
25. ed on next page Construction of n Vitro Multimers Continued Materials Needed You will the following items e Electrocompetent or chemically competent E coli must be recA endA for transformation page v You will need 3 4 tubes of competent cells per experiment e BamH I and Bgl II restriction enzymes and appropriate buffers e Low melt agarose gel e PureLink Quick Gel Extraction Kit or S N A P Gel Purification Kit page v or glass milk e Sterile water e CIAP calf intestinal alkaline phosphatase 1 unit ul page v e 10X CIAP Buffer supplied with CIAP page v e Phenol chloroform e 3M sodium acetate e 100 ethanol e 80 ethanol e T4 Ligase 2 5 units ul page v e 10X Ligation Buffer with ATP e Low Salt LB plates containing 25 ug ml Zeocin page 17 e 150mm 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 pPICZ the parent vector and pPICZ 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
26. eptide 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 If you wish to express your protein without the C terminal peptide be sure to include a stop codon pPICZ A B and C contain unique Bgl II and BamH I sites to allow construction of plasmids containing multiple copies of your gene For information on how to construct multimers refer to pages 24 31 Continued on next page Cloning into pPICZ A B and C Continued Multiple Cloning Below is the multiple cloning site for pPICZ A Restriction sites are labeled to Site of pPICZ A indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing You can download the complete sequence of pPICZ A from our web site at www invitrogen com or by contacting Technical Support see page 32 For a map and a description of the features of pPICZ refer to the Appendix pages 21 22 5 end of AOX1 mRNA 5 AOX1 priming site 811 AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA xx 871 CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT Sfu EcoR Pml Sfi BsmB Asp718 Kpn I Xho 931 ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC Sa
27. es 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 T and Hashimoto Gotoh T 1988 Tandem Gene Amplification in vitro for Rapid and Efficient Expression in Animal Cells Gere 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 31 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 web site 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 Fou
28. gs 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 CooHssN2102 3 and the molecular weight is 1 535 The diagram below shows the structure of Zeocin MW 1 535 Zeocin is used for selection in mammalian cells Mulsant ef 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 Organism Zeocin Concentration and Selective Medium E coli 25 50 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 Zeocln Continued Handling Zeocin 20 TM High salt and acidity or basicity inactivate Zeocin therefore we
29. heir 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 warranty 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 Purchaser Notification Introduction Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 74 Pichia Pastoris Expression System The Pichia Expression System is covered under the licenses detailed below 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 Swit
30. in press Henikoff S and Cohen E H 1984 Sequences Responsible for Transcription Termination on a Gene Segment in Saccharomyces cerevisiae Mol Cell Biol 4 1515 1520 Higgins D R and Cregg J M eds 1998 Pichia Protocols Vol 103 Methods in Molecular Biology Edited by Walker J M 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 Romanos M A Scorer C A and Clare J J 1992 Foreign Gene Expression in Yeast A Review Yeast 8 423 488 Sambrook J Fritsch E F and Maniatis T 1989 Molecul
31. ion 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 Expression 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 V
32. le at 15 psi and 121 C for 20 minutes 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 Sterile water 2 agar Optional If making YPD slants or plates 2 dextrose glucose Zeocin in appropriate concentration 1 2 3 4 5 Dissolve 10 g 1 yeast extract and 20 g 2 peptone in 900 ml water Optional Add 20 g of 2 agar if making YPD slants or plates Dissolve Autoclave for 20 minutes on liquid cycle Add 100 ml of 2 dextrose filter sterilize dextrose before use Cool solution to 60 C and add the appropriate amount of Zeocin from a 100 mg ml stock solution 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 TM Store YPD slants or plates containing Zeocin at 4 C The shelf life is 1 2 weeks Continued on next page 17 Recipes Continued YPDS Zeocin Agar Breaking Buffer 18 Yeast Extract Peptone Dextrose Medium with Sorbitol 1 liter 1 yeast extract 2 peptone 1 M sorbitol 2 agar Sterile water 2 dextrose glucose Zeocin in appropriate concentration 1 Dissolve the following item in 900 ml water e 10 g y
33. low 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 1 M 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 200 pl on 150 mm plates containing the appropriate concentration of TM Zeocin 5 Optional Continue 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 pl on TM 150 mm plates containing the appropriate concentration of Zeocin 6 Incubate plates for 2 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 pPICZ 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 amp Cregg 1998 You are now ready to test yo
34. 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 BamH I linearized recombinant pPICZ 1 Setup a 20 ul ligation reactions as follows e 15 ul Bgl II BamH I digested expression cassette e 2ylsterile water e 2 ul 10X Ligation Buffer with ATP 1ulT4 DNA Ligase 2 5 units ul 2 Incubate at 16 C for 2 5 hours Heat inactivate the ligase by incubating at 65 C for 20 minutes d 4 Addthe following reagents for restriction enzyme digestion cut back Note BamH I and Bgl II may be used with the same reaction buffer e 23 pl sterile water e 5yl 10X restriction enzyme buffer e Lui Bgl II 10 units pl e Lu BamHI 10 units ul 5 Incubate the reaction at 37 C for 2 hours 6 Add 50 ul phenol chloroform and extract the restriction enzyme digestion to remove the enzymes Transfer the aqueous solution to a new microcentrifuge tube 7 Add 5 unl of 3M sodium acetate and 110 ul of 100 ethanol to ethanol precipitate the DNA 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
35. nologies 101 North Wilmot Road Suite 600 Tucson Arizona 85711 3335 Tel 520 748 4443 Fax 520 748 0025 33 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 Greene Publishing Associates and Wiley Interscience New York 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 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 Nucleic 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 L 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 in Methods in Molecular and Cellular Biology
36. ntain 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 32 Safety Data Sheets SDSs are available at www invitrogen com sds The Certificate 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 t
37. or Enzyme Restriction Site bp Supplier Sac I 209 Many Pure 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 4 Centrifuge 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 Preparation of Pichia for Electroporation Transformation by Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation 1 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 4 C sterile water Centrifuge the cells as in Step 3 then resuspend the pellet with 250 ml of ice cold 0 4 C sterile water Centrifuge the cells as in Step 3 then resuspen
38. provide a signal to normalize your data Continued on next page 25 Construction of n Vitro Multimers Continued Important Digestion of Recombinant pPICZ Production of Expression Cassettes for Multimerization 26 Once you have created a pPICZ plasmid containing multimers note that this 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 pg of DNA to compensate for the 10 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 e Plate out all of the transformation mix on several YPDS plates containing the appropriate concentration of Zeocin You will need to use the optional outgrowth procedure on page 10 Set up two separate digests of recombinant pPICZ containing one copy of your gene 1 Double digest 1 2 ug of recombinant pPICZ in 20 ul with 10 units each of Bgl II and BamH I Proceed to Production of Expression Cassettes for Multimerization Step 1 2 Digest 2 ng of recombinant pPICZ in 20 ul with 10 units of BamH I only Proceed to Dephosphorylation of Vector Step 1 TM The S N A P Gel Purification Kit available
39. ropagate pPICZ A B and C by transformation into a recA endA1 E coli strain such as TOP10 DH5 or JM109 2 Develop a cloning strategy and ligate your gene into one of the pPICZ vectors in frame with the C terminal tag 3 Transform into E coli and select transformants on Low Salt LB plates containing 25 pg ml Zeocin 4 Analyze 10 20 transformants by restriction mapping or sequencing to confirm in frame fusion of your gene with the C terminal tag 5 Purify and linearize the recombinant plasmid for transformation into Pichia pastoris 6 Transform your Pichia strain and plate onto YPDS plates containing the appropriate concentration of Zeocin 7 Select for Zeocin resistant transformants 8 Optimize expression of your gene 9 Purify your fusion protein on metal chelating resin i e ProBond Continued on next page Methods Cloning into pPICZ A B and C Introduction General Molecular Biology Techniques E coli Strain Transformation Method Maintenance of Plasmids The pPICZ 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 C terminal peptide General considerations for cloning and transformation are discu
40. s are available from Invitrogen The NuPAGE Gel System avoids the protein modifications associated with Laemmli type SDS PAGE ensuring optimal separation for protein analysis 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 visit our web site at www invitrogen com or contact Technical Support page 32 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 x for ordering information or an antibody to your protein of interest In addition the Positope Control Protein page v is available from Invitrogen for use as a positive control for detection of fusion proteins containing a c myc epitope or a polyhistidine 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 visit our web site at www invitrogen com or contact Technical Support page 32 Because the pPICZ 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
41. sate with 7 ml of the Denaturing Binding Buffer TM 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 specific 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 Kit before adding it to the column The pH should be greater than or equal to 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 Low Salt LB Medium with Zeocin YPD Zeocin Appendix 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 1N NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cyc
42. sformation next page Pichia Transformation Introduction Zeocin Selection Method of Transformation Pichia EasyComp Transformation Kit Important You should now have your gene cloned into one of the pPICZ vectors Your construct should be correctly fused to the C terminal peptide if desired This section provides general guidelines to prepare plasmid DNA transform your Pichia strain and select for Zeocin resistant clones TM We generally use 100 pg ml Zeocin to select for transformants when using the X 33 Pichia strain If you are transforming your pPICZ 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 TM containing Zeocin does not yield any transformants We recommend electroporation for transformation of Pichia with pPICZ A B or C Electroporation yields 10 to 10 transformants per ug of linearized DNA and does not destroy the cell wall of Pichia
43. ssed 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 pPICZ vectors including TOP10 JM109 and DH5 We recommend that you propagate the pPICZ 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 page v 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 pPICZ vectors contain the Zeocin resistance Sh ble gene to allow selection of the plasmid using Zeocin To propagate and maintain the pPICZ plasmids we recommend using the following procedure 1 Use 10 ng of your vector to transform a recA endA E coli strain like TOP10 DH5 JM109 or equivalent see above 2 Select transformants on Low Salt LB plates containing 25 pg 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 pPICZ
44. tab C181 00 KM71H Pichia strain 1 stab C182 00 SMD1168H Pichia strain 1 stab C184 00 pPICZa A B and C 20 ug each V195 20 pPIC6a A B and C 20 ug each V215 20 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 PureLink Gel Extraction Kit 50 preps K2100 12 250 preps K2100 25 S N A P Gel Purification Kit 25 preps K1999 25 PureLink Quick Plasmid Miniprep Kit 50 preps K2100 10 250 preps K2100 11 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 50 preps K2100 13 One Shot TOP10 chemically competent E coli 10 reactions C4040 10 20 reactions C4040 03 One Shot TOP10 Electrocompetent E Coli 10 reactions C4040 50 20 reactions C4040 52 TOP10 Electrocomp Kits 20 reactions C664 55 Positope Control Protein 5ug R900 50 CIAP Calf Intestinal Alkaline Phosphatase 1 000 units 18009 019 T4 DNA Ligase 100 units 15224 017 500 units 15224 025 Zeocin 1g R250 01 5g R250 05 p Gal Assay Kit 1 kit K1455 01 B Gal Staining Kit 1 kit K1465 01 E Gel Agarose Gels are bufferless pre cast agarose gels designed for fast convenient electrophoresis of DNA samples E Gel agarose gels are available in different agarose percentage and well format for your convenience For more details on these products visit our web site at www
45. te that Bgl II and BamH I 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 BamH I and Bgl II sites 5 Treat the ligation mix with BamH I and Bg II to eliminate head to head and tail to tail multimers 6 Ligate into BamH I linearized recombinant pPICZ Transform into E coli and analyze recombinant plasmids for copy number by digesting with Bgl II and BamH I 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 pPICZ A B or C For example Stage Description 1 Digest pPICZ containing one copy of your gene with BamH I 2 Ligate a single copy of the Bg II BamH I expression cassette into BamH I 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 BamH I and Bg II to release a cassette with 2 copies of your gene optional 5 Digest the vector with 2 copies of your gene with BamH I 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 24 Continu
46. ter transformation into E coli CIAP defective Use fresh CIAP 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 BamH I Bgl II 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 ligating each expression cassette stepwise see page 28 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 is low Transform using more DNA and or transformants do multiple transformations with more DNA and cells For More There are a number references in the literature you can consult in order to optimize Information 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 BioTechniqu
47. the order given to the cells PEG shields the cells from the detrimental effects of the high LiCl concentration i 240 ul50 PEG ii 36 ul 1 M LiCl iii 25 pl 2 mg ml single stranded DNA iv 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 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 25 100 pl on YPD plates containing the TM appropriate concentration of Zeocin Incubate the plates for 2 3 days at 30 C Do SS Oy rows 23 Construction of n Vitro Multimers Experimental At this point you should have your gene cloned into the multiple cloning site of Outline either pPICZ A B or C To generate multiple copies of your expression cassette Stage Description 1 Digest pPICZ containing your gene of interest with Bgl Il and BamH I to release the expression cassette Paox plus your gene 2 To clone multiple copies of the expression cassette linearize pPICZ containing your gene of interest using BamH I Note that the BamH I linearized vector already contains one copy of your expression cassette 3 Treat the Bgl II BamH I expression cassette with ligase in vitro No
48. tidine tag bases 1057 1077 Apa in pPICZA 3 AOX priming site bases 1159 1179 Xba in pPICZ B 3 end of MRNA base 1250 SnaB in pPICZ C AOX7 transcription termination region bases 1078 1418 Fragment containing TEF1 promoter bases 1419 1830 EM7 promoter bases 1831 1898 Sh ble ORF bases 1899 2273 CYC1 transcription termination region bases 2274 2591 pUC origin bases 2602 3275 complementary strand Continued on next page 21 Map and Features of pPICZ A B and C Continued Features of pPICZ pPICZ A 3329 bp pPICZ B 3328 bp and pPICZ C 3329 bp contain the A B and C following elements All features have been functionally tested Feature Benefit 5 AOXI 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 AOX1 locus Multiple cloning site Allows insertion of your gene into the expression vector c myc epitope Glu Gln Lys Leu lle Ser Glu Glu Asp Leu Permits detection of your recombinant fusion protein with the Anti myc Antibody or Anti myc HRP Antibody see page vi for ordering information Evans et al 1985 C terminal polyhistidine 6xHis tag Permits purification of your recombinant fusion TM 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 page
49. to ligate your insert immediately or you can store at 20 C Proceed to Ligation of Multimers into Linearized Vector 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 restriction enzymes and add 1 mM ATP to the reaction in order 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 Continued on next page Construction of n Vitro Multimers Continued Ligation of Multimers into Linearized Vector Transformation into E coli You are now ready to ligate the mixture of multimers generated in Step 10 above into dephosphorylated linearized vector 1 2 3 Set up the following ligation reactions Dephosphorylated vector page 27 Step 10 4 ul Expression cassette multimers Step 10 above 4 ul 10X Ligation Buffer 1 ul T4 DNA Ligase 2 5 units ul lul 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 yl Total volume 10 pl Incubate overnight at 16 C You can store the ligation reactions at 20 C until ready to use or transform 1 10 ul of each ligation mix into competent E coli Note
50. uct Restriction Digest 10 Host strains containing the his4 allele e g GS115 and transformed with the pPICZ vectors require histidine when grown in minimal media Add histidine to a final concentration of 0 004 to ensure growth of your transformants The pPICZ vectors do not contain a yeast origin of replication Transformants can only be isolated if recombination occurs between the plasmid and the Pichia genome You will need the following items Note Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear to be somewhat osmotically sensitive e 5 10 pg pure pPICZ 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 TM e YPDS plates containing the appropriate concentration of Zeocin see page 18 for recipe To promote integration we recommend that you linearize your pPICZ construct within the 5 AOXI region The table below lists unique sites that may be used to linearize pPICZ 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 pPICZ Be sure that your insert does not contain the restriction site you wish to use to linearize your vect
51. ude Bgl II BamH I digested pPICZ as a control It is possible to get vector rearrangements and deletions with large recombinant vectors in E coli Including Bg II BamH I digested pPICZ 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 Note 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 the outgrowth protocol on page 10 to isolate transformants Continued on next page 30 Construction of n Vitro Multimers Continued Troubleshooting Pichia The table below will help you optimize formation and isolation of multimers in Problem Cause Solution No multimers or low number of multimers in your vector af
52. ur transformants for expression of your gene of interest See Expression in Pichia next page Expression in Pichia Introduction Control Strain Note Detection of 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 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 As a positive control for expression GS115 pPICZ lacZ is provided For expression use the small scale Mut protocol described in the Pichia Expression System manual Expression in shake flasks is detectable after 48 hours and reaches the maximum at 96 hours 4 days galactosidase is detected using SDS PAGE and staining the gel with Coomassie Blue or the ONPG assay f Gal Assay page v Cells expressing f galactosidase can be detected by plating on medium containing methanol and X gal Note that once you have obtained Zeocin resistant transformants it is not necessary to maintain your re
53. vi Lindner et al 1997 and the Anti His C term HRP Antibody page vi 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 numbers D12478 D01130 Transcription elongation factor 1 gene promoter from Saccharomyces cerevisiae that drives expression of the TM 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 number M34014 3 end of the Saccharomyces cerevisiae CYC1 gene that allows efficient 3 mRNA processing of the Zeocin resistance gene for increased stability pUC origin Allows replication and maintenance of the plasmid in E coli 27 Lithium Chloride Transformation Method Introduction Preparation of Solutions Preparation of Cells Transformation This is a modified version of the procedure described for S cerevisiae Gietz amp Schiestl 1996 and is provided as an alternative to transformation by electroporation Transformation efficiency is between 10 to 10 cfu ug linearized DNA
54. zerland 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 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 Express

pPICZ A, B, and C - Thermo Fisher Scientific

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