pBAD/TOPO® ThioFusion™ Expression Kit
Contents
1. 6 Analyze by agarose gel electrophoresis Important If you have problems obtaining transformants or the correct insert see pages 25 26 Control reactions are described using reagents supplied in the kit 10 Continued on next page TOPO Cloning Reaction and Transformation Continued Long Term Storage After you have identified the correct clone purify the colony and make a glycerol stock for long term storage We recommend that you also store a stock of plasmid DNA at 20 C 1 Streak the original colony out for single colony on LB plates containing 50 100 pg mL ampicillin 2 Isolate a single colony and inoculate into 1 2 mL of LB containing 50 100 ug mL ampicillin 3 Grow overnight until culture is saturated 4 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store glycerol stock at 80 C and a stock of plasmid DNA at 20 C 11 Optimizing the TOPO Cloning Reaction Introduction Faster Subcloning More Transformants Cloning Dilute PCR Products 12 The information below will help you optimize the TOPO Cloning reaction for your particular needs The high efficiency of TOPO Cloning technology allows you to streamLine the cloning process If you routinely clone PCR products and wish to speed up the process consider the following e Incubate the TOPO Cloning reaction for only 30 seconds instead of 5 minutes You may not obtain the highest2. C 1X 3 Remove 10 uL from the reaction and analyze by agarose gel electrophoresis A discrete 500 bp band should be visible Proceed to the Control TOPO Cloning Reactions next page Continued on next page 25 pBAD TOPO ThioFusion Control Reactions Continued Control TOPO Cloning Reactions Analyzing Results Transformation Control 26 Using the control PCR product produced on the previous page and the pBAD Thio TOPO vector set up two 6 uL TOPO Cloning reactions as described below 1 Setup control TOPO Cloning reactions Reagent Vector Only Vector PCR Insert Control PCR Product Tub Salt Solution or Dilute Salt Tub 1uL Solution Sterile Water 4 ul 3 pL pBAD Thio TOPO 1 pL 1 pL Incubate at room temperature for 5 minutes and place on ice Transform 2 pL of each reaction into separate vials of TOP10 One Shot cells page 9 4 Spread 10 50 uL of each transformation mix onto LB plates containing 50 100 ug mL ampicillin and X Gal see page 20 Plate two different volumes to ensure that at least one plate has well spaced colonies To plate small volumes add 20 uL of SOC to allow even spreading 5 Incubate overnight at 37 C The vector PCR insert reaction should produce hundreds of colonies and greater than 90 of the colonies should be blue The vector only plate should yield very few colonies 1076 of the vector PCR insert pla
3. Remember that your PCR product will have single 3 adenine overhangs If you wish to Then clone in frame with thioredoxin the forward PCR primer must be designed to ensure that your protein is in frame with the N terminal leader peptide include the V5 epitope and polyhistidine region the reverse PCR primer must be designed to remove the native stop codon in the gene of interest and preserve the reading frame through the C terminal tag NOT include the V5 epitope and polyhistidine include the native sequence containing the stop codon region in the reverse primer or make sure the stop codon is upstream from the reverse PCR primer binding site remove the N terminal leader for expressing the forward PCR primer can be designed to include a truly native protein unique Nco I site which contains the first ATG of the Note Removing the N terminal leader generally protein decreases expression levels Example 5 ACC ATG G The vector can be digested with Nco I after cloning and religated assuming there are no internal Nco I sites in your PCR product When synthesizing PCR primers do not add 5 phosphates to the primers because 5 phosphates prevent the synthesized PCR product from ligating into Net the pBAD TOPO vector Continued on next page Designing PCR Primers Continued TOPO Cloning Site 81 16 24 32 39 457 523 589 655 721 787 861
4. 2 Add water to 1 liter and autoclave for 20 minutes on the liquid cycle 3 Store at room temperature 50 mM potassium phosphate pH 7 8 400 mM NaCl 100 mM KCl 10 glycerol 0 5 Triton X 100 10 mM imidazole 1 Prepare 1 M stock solutions of KH PO and KoHPO 2 For 100 mL dissolve the following reagents in 90 mL of deionized water 1M KH PO 0 3 mL 1M KHPO 4 7 mL NaCl 23g KCI 0 75 g Glycerol 10 mL Triton X 100 0 5 mL Imidazole 68 mg 3 Mix thoroughly and adjust pH to 7 8 with HCL Bring the volume to 100 mL 4 Store at 4 C 21 Purifying the PCR Products Introduction Using the PureLink Quick Gel Extraction Kit 22 Smearing multiple banding primer dimer artifacts or large PCR products gt 3 kb may necessitate gel purification If you intend to purify your PCR product be extremely careful to remove all sources of nuclease contamination There are many protocols to isolate DNA fragments or remove oligonucleotides Refer to Current Protocols in Molecular Biology Unit 2 6 Ausubel et al 1994 for the most common protocols Two simple protocols are described below TM The PureLink Quick Gel Extraction Kit allows you to rapidly purify PCR 10 11 12 13 products from regular agarose gels see page 32 for ordering information Equilibrate a water bath or heat block to 50 C Cut the area of the gel containing the desired DNA fragment using a clean sharp blade Minimize t
5. 941 1021 The diagram below is supplied to help you design appropriate PCR primers to correctly clone and express your PCR product Restriction sites are labeled to indicate the actual cleavage site The complete sequence of the vector is available for downloading at www invitrogen com or from Technical Support page 34 O7 Region AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA O Region pBAD Forward priming site CAP binding site I CGGCAGAAAA GTCCACATTG ATTATTTGCA CGGCGTCACA CTTTGCTATG CCATAGCATT TTTATCCATA AGATTAGCGG L I and I Region 35 10 TERR Pl ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTTGGG CTAGAAATAA TTTTGTTTAA RBS NcoI HP Thioredoxin translational start site l l CTTTAAGAAG GAGATATACA TACCC ATG Met ACT GAT GTA CTT AAG GCA GAT GGT Thr Asp Val Leu Lys Ala Asp Gly AAA ATG ATC GCT CCG ATT CTG GAT Lys Met Ile Ala Pro Ile Leu Asp CTG AAC ATC GAT CAC AAC CCG GGC Leu Asn Ile Asp His Asn Pro Gly CTG TTC AAA AAC GGT GAA GTG GCG Leu Phe Lys Asn Gly Glu Val Ala Trx Forward priming site NgoM I Nae I f je E a TTC CTC GAC GCT AAC CTG GCC GGC Phe Leu Asp Ala Asn Leu Ala Gly GGA TCT GAT AAA ATT ATT CAT CTG ACT GAT GAT Gly Ser Asp Lys Ile Ile His Leu Thr Asp Asp HP Thioredoxin ORF indicated by italicized amino acids G
6. Expression System is licensed under U S Patent No 5 270 181 License No 29 from Genetics Institute Inc for research use only Licenses for Thiofusion commercial manufacture or use may be obtained directly from Genetics Institute Expression Inc 87 Cambridgepark Drive Cambridge MA 02140 System Information for The LMG194 cell line is genetically modified As a condition of sale this product European must be in accordance with all applicable local legislation and guidelines Customers including EC Directive 90 219 EEC on the contained use of genetically modified organisms 36 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 Brownstein M J Carpten J D and Smith J R 1996 Modulation of Non Templated Nucleotide Addition by Taq DNA Polymerase Primer Modifications that Facilitate Genotyping BioTechniques 20 1004 1010 Carson M J Barondess J J and Beckwith J 1991 The FtsQ Protein of Escherichia coli Membrane Topology Abundance and Cell Division Phenotypes Due to Overproduction and Insertion Mutations J Bacteriol 173 2187 2195 Dalbey R E and Wickner W 1985 Leader Peptidase Catalyzes the Release of Exported Proteins from the Outer Surface of the Escherichia coli Plasma Membrane J Biol Chem 260 15925 15
7. Once you have decided on a PCR strategy and have synthesized the primers you are ready to produce your PCR product Materials Supplied Taq polymerase by the User Note For improved specificity and higher yields we recommend using Platinum Taq DNA Polymerase available from Invitrogen see page 32 for ordering information to generate your PCR product e Thermocycler e DNA template and primers to produce your PCR product Note dNTPs adjusted to pH 8 are provided in the kit Polymerase If you wish to use a mixture containing Tag polymerase and a proofreading Mixtures polymerase Taq must be used in excess of a 10 1 ratio to ensure the presence of 3 A overhangs on the PCR product If you use polymerase mixtures that do not have enough Tag polymerase or a proofreading polymerase only you can add 3 A overhangs using the method on page 24 Producing PCR 1 Set up the following 50 uL PCR reaction Use less DNA if you are using Products plasmid DNA as a template and more DNA if you are using genomic DNA as a template Use the cycling parameters suitable for your primers and template Be sure to include a 7 to 30 minute extension at 72 C after the last cycle to ensure that all PCR products are full length and 3 adenylated DNA Template 10 100 ng 10X PCR Buffer 5 pL 50 mM dNTPs 0 5 pL Primers 100 200 ng each Sterile water add to a final volume of 49 uL Tag Polymerase 1 unit uL lul Total Volume 50 uL 2 Check the
8. PCR product by agarose gel electrophoresis You should see a single discrete band If you do not see a single band refer to the Note below If you do not obtain a single discrete band from your PCR gel purify your fragment before TOPO Cloning see page 22 Take special care to avoid Note a sources of nuclease contamination and long exposure to UV light Alternatively optimize your PCR to eliminate multiple bands and smearing Innis et al 1990 The PCR Optimizer Kit from Invitrogen can help you optimize your PCR see page 32 for ordering information TOPO Cloning Reaction and Transformation Introduction Note Important Chemically Competent E coli Electrocompetent E coli Materials Supplied by the User TOPO Cloning technology allows you to produce your PCR products ligate them into pBAD Thio TOPO and transform the recombinant vector into E coli in one day It is important to have everything ready to use to ensure you obtain the best possible results If this is the first time you have TOPO Cloned perform the control reactions on pages 25 26 in parallel with your samples Experiments at Invitrogen demonstrate that inclusion of salt 200 mM NaCl 10 mM MgCl in the TOPO Cloning reaction increases the number of transformants 2 to 3 fold We have also observed that in the presence of salt incubation times of greater than 5 minutes can also increase the number of transformants This is
9. for protocol 2 To optimize expression of your protein try arabinose concentrations spanning the amount determined in Step 1 or you may perform a time course Note If your protein is insoluble analyze the supernatant and the pellet of lysed cells for expression of soluble protein page 16 Expressing your protein with the N terminal HP thioredoxin peptide and the C terminal tag increases the size of your protein by 13 kDa and 3 kDa respectively Be sure to account for any additional amino acids between the tag and your protein Continued on next page 13 Expressing the PCR Product Continued Materials Required Pilot Expression 14 SOB or LB containing 50 100 ug mL ampicillin see page 19 for recipe 37 C shaking incubator 20 L arabinose provided Additional L arabinose is available from Sigma Cat no A3256 In addition to testing your transformants we recommend that you include the pBAD Thio vector as a positive control and cells without vector as a negative control 1 For each transformant or control inoculate 2 mL of SOB or LB containing 50 100 ug mL ampicillin with a single recombinant E coli colony Note If you are using LMG194 as a host use RM medium containing glucose and 100 ug mL ampicillin for overnight growth see page 21 for a recipe and then substitute glycerol for glucose in medium at Step 3 below see Using LMG194 previous page Grow overnight at 37 C with shaking
10. in contrast to earlier experiments without salt where the number of transformants decreases as the incubation time 5 minutes Including salt in the TOPO Cloning reaction allows for longer incubation times because it prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA The result is more intact molecules leading to higher transformation efficiencies We recommend adding salt to the TOPO Cloning reaction A stock salt solution is provided in the kit for this purpose Note that the amount of salt added to the TOPO Cloning reaction varies depending on whether you plan to transform chemically competent cells provided or electrocompetent cells see below For this reason two different TOPO Cloning reaction protocols are provided to help you obtain the best possible results For TOPO Cloning and transformation into chemically competent E coli adding NaCl and MgCb to a final concentration of 200 mM NaCl 10 mM MgCl in the TOPO Cloning reaction increases the number of colonies over time A Salt Solution 1 2 M NaCl 0 06 M MgCl is provided to adjust the TOPO Cloning reaction to the recommended concentration of NaCl and MgCl For TOPO Cloning and transformation of electrocompetent E coli salt must also be included in the TOPO Cloning reaction but the amount of salt must be reduced to 50 mM NaCl 2 5 mM MgCl to prevent arcing The Salt Solut
11. irradiated 200 mg 11593 027 B Gal Antiserum 50 uL R901 25 B Gal Assay Kit 100 reactions K1455 01 B Gal Staining Kit 1 kit K1465 01 X gal 100 mg 15520 034 Electrocompetent TOP10 cells are also available as electrocompetent cells See the table below for Cells ordering information Kit Reactions Cat no One Shot TOP10 Electrocomp E coli 10 C4040 50 20 C4040 52 TOP10 Electrocomp Kits 20 C664 55 40 C664 11 120 C664 24 Continued on next page 32 Accessory Products Continued Detecting Recombinant Protein Purifying Recombinant Protein Important Expression of your recombinant fusion protein can be detected using an antibody to the protein itself or to the appropriate epitope The table below describes the antibodies available for use with pBAD Thio TOPO Horseradish peroxidase HRP conjugated antibodies allow one step detection using colorimetric or chemiluminescent detection methods Epitope Antibody Cat no V5 Anti V5 R960 25 CGKPIPNPLEGLDST Anti V5 HRP R961 25 C terminal polyhistidine tag Anti His C term R930 25 CHHHHHH COOH Anti His C term HRP R931 25 Thioredoxin non contiguous epitope Anti Thio R920 25 The metal binding domain encoded by either the His Patch thioredoxin or the 6xHis tag allows simple easy purification of your recombinant fusion protein by Immobilized Metal Affinity Chromatography IMAC us
12. number of colonies but with the high cloning efficiency of TOPO Cloning most of the transformants will contain your insert e After adding 2 uL of the TOPO Cloning reaction to chemically competent cells incubate on ice for only 5 minutes Increasing the incubation time to 30 minutes does not significantly improve transformation efficiency If you are TOPO Cloning large PCR products toxic genes or cloning a pool of PCR products you may need more transformants to obtain the clones you want To increase the number of colonies incubate the salt supplemented TOPO Cloning reaction for 20 to 30 minutes instead of 5 minutes Increasing the incubation time of the salt supplemented TOPO Cloning reaction allows more molecules to ligate increasing the transformation efficiency Addition of salt appears to prevent topoisomerase from rebinding and nicking the DNA after it has ligated the PCR product and dissociated from the DNA To clone dilute PCR products you may e Increase the amount of the PCR product e Incubate the TOPO Cloning reaction for 20 to 30 minutes e Concentrate the PCR product Expressing the PCR Product Introduction LMG194 Strain pBAD Thio Vector Basic Strategy Note Since each recombinant protein has different characteristics that may affect optimal expression it is helpful to vary the arabinose concentration and or run a time course of expression to optimize the expression of your particular p
13. the 3 phosphate of the cleaved strand and a tyrosyl residue Tyr 274 of topoisomerase I The phospho tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5 hydroxyl of the original cleaved strand reversing the reaction and releasing topoisomerase Shuman 1994 Topoisomerase poate FNAGGG GGGA Uu A PCR Product noe HO S Topoisomerase Continued on next page Description of the System Continued Regulation of Expression by Arabinose Thioredoxin His Patch Thioredoxin In the presence of arabinose expression from Psap is induced while only very low levels of transcription are observed from Psap in the absence of arabinose Lee 1980 Lee et al 1987 Uninduced levels are repressed even further by growth in the presence of glucose 0 1 to 0 2 Glucose reduces the levels of 3 5 cyclic AMP lowering expression from the catabolite repressed Pgap promoter Miyada et al 1984 By varying the concentration of arabinose protein expression levels can be optimized to ensure maximum expression of protein In addition the tight regulation of Paap by AraC is useful for expression of potentially toxic or essential genes Carson et al 1991 Dalbey and Wickner 1985 Guzman et al 1992 Kuhn and Wickner 1985 Russell et al 1989 San Millan et al 1989 For more information on the mechanism of expression and repression of the ara regulon see page 30 or refer to Schleif 1992 Th
14. transformation on a prewarmed selective plate and incubate overnight at 37 C To ensure even spreading of small volumes add 20 uL of SOC We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies An efficient TOPO Cloning reaction will produce hundreds of colonies Pick 10 colonies for analysis see Analyzing Positive Clones next page Adding the Dilute Salt Solution in the TOPO Cloning Reaction brings the final concentration of NaCl and MgCl in the TOPO Cloning reaction to 50 mM and 2 5 mM respectively To prevent arcing of your samples during electroporation the volume of cells should be between 50 and 80 uL for 0 1 cm cuvettes or 100 to 200 uL for 0 2 cm cuvettes If you experience arcing during transformation try one of the following Reduce the voltage normally used to charge your electroporator by 10 Reduce the pulse length by reducing the load resistance to 100 ohms Ethanol precipitate the TOPO Cloning reaction and resuspend in water prior to electroporation Continued on next page TOPO Cloning Reaction and Transformation Continued Analyzing Positive 1 Clones 2 Culture 10 colonies overnight in LB or SOB medium with 50 100 pg mL ampicillin Isolate plasmid DNA using your method of choice If you need ultra pure plasmid DNA for automated or manual sequencing we recommend using Invitrogen s PureLink HO Mini Plasmid Purification or PureLink HiP
15. 225 250 rpm to ODeoo 1 2 The next day label five tubes 1 through 5 and add 10 mL of SOB or LB containing 50 100 ug mL ampicillin Inoculate each tube with 0 1 mL of the overnight culture Grow the cultures at 37 C with vigorous shaking to an OD 0 5 the cells should be in mid log phase While the cells are growing prepare four 10 fold serial dilutions of 2076 arabinose with sterile water using aseptic technique e g 276 0 276 0 0276 and 0 002 Remove a 1 mL aliquot of cells from each tube centrifuge at maximum speed in a microcentrifuge for 30 seconds and aspirate the supernatant Freeze the cell pellet at 20 C This is the zero time point sample protocol continued on next page Continued on next page Expressing the PCR Product Continued Pilot Expression continued Expressing Toxic Proteins 9 Use the stock solutions prepared in Step 6 and add arabinose to the five 9 mL cultures as follows Note For the positive and negative controls it is not necessary to test all concentrations of arabinose Use only the highest concentration of arabinose Tube Stock Solution Volume mL Final Concentration 1 0 002 0 09 0 00002 2 0 02 0 09 0 0002 3 0 2 0 09 0 002 4 2 0 09 0 02 5 20 0 09 0 2 10 Grow at 37 C with shaking for 4 hours 11 Take 1 mL samples at 4 hours and treat as in Step 7 and 8 You will have a total of 10 samples for each trans
16. 5 minutes Protect plates from light 1 Fora40 mg mL stock solution dissolve 400 mg X Gal in 10 mL dimethylformamide 2 Protect from light by storing in a brown bottle at 20 C 2 Tryptone 0 5 Yeast Extract 0 05 NaCl 2 5 mM KCI 10 mM MgCl 1 Dissolve 20 g tryptone 5 g yeast extract and 0 5 g NaCl in 950 mL deionized water 2 Makea250 mM KCI solution by dissolving 1 86 g of KCI in 100 mL of deionized water Add 10 mL of this stock KCI solution to the solution in Step 1 3 Adjust pH to 7 5 with 5 M NaOH and add deionized water to 1 liter Autoclave this solution cool to 55 C and add 10 mL of sterile 1 M MgCl You may also add ampicillin to 50 100 pg mL 5 Store at 4 C Medium is stable for only 1 2 weeks Continued on next page Recipes Continued RM Medium Glucose 10X M9 Salts Lysis Buffer 1X M9 Salts see recipe below for 10X M9 Salts 2 Casamino Acids 0 2 glucose 1mM MgCl 50 100 pg mL ampicillin 1 For 1 liter of RM medium mix 20 g Casamino Acids and 890 mL deionized water 2 Autoclave 20 minutes on liquid cycle 3 Cool the autoclaved solution and add the following sterile solutions 10X M9 Salts 100 mL 1M MgCl 1 mL 20 glucose 10 mL 100 mg mL ampicillin 0 5 to 1 mL 4 Mix well and store at 4 C for 1 month 1 Dissolve the following reagents in 900 mL water and adjust the pH to 7 4 witt 10 M NaOH Na HPO 60g KH PO 30 8 NaCl 5g NH4CI 10g
17. 672 Enterokinase recognition site bases 691 705 TOPO Cloning site bases 714 715 V5 epitope bases 730 771 Polyhistidine region bases 781 801 pBAD Reverse priming site bases 854 871 rrnB transcriptional termination region bases 904 1061 Ampicillin resistance gene ORF bases 1341 2201 pUC origin bases 2346 3019 AraC ORF bases 3550 4428 opposite strand Continued on next page 28 Map and Features of pBAD Thio TOPO Continued Features of The important elements of pBAD Thio TOPO 4 454 bp are described in the pBAD T hio TOPO following table All features have been functionally tested For more information on the regulation of gene expression by arabinose see page 31 Feature Benefit araBAD promoter Paap Provides tight dose dependent regulation of heterologous gene expression Guzman et al 1995 O region Binding site of AraC that represses transcription from Pran O region Binding site of AraC that represses transcription of the araC promoter Pc transcribed on the opposite strand CAP binding site Site where CAP cAMP binding protein binds to activate transcription from Paap and Pc I and I regions Binding sites of AraC that activate transcription from Psap 10 and 35 regions Binding sites of RNA polymerase for transcription from Paap Optimized ribosome binding site Increases efficiency of recombinant fusion protein expression HP thio
18. 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
19. 931 Deutscher M P 1990 Guide to Protein Purification In Methods in Enzymology Vol 182 J N Abelson and M I Simon eds Academic Press San Diego CA Guzman L M Barondess J J and Beckwith J 1992 FtsL an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli J Bacteriol 174 7716 7728 Guzman L M Belin D Carson M J and Beckwith J 1995 Tight Regulation Modulation and High Level Expression by Vectors Containing the Arabinose Pp ap Promoter J Bacteriol 177 4121 4130 Holmgren A 1985 Thioredoxin Ann Rev Biochem 54 237 271 Innis M A Gelfand D H Sninsky J J and White T S 1990 PCR Protocols A Guide to Methods and Applications Academic Press San Diego CA Katti S K LeMaster D M and Eklund H 1990 Crystal Structure of Thioredoxin from E coli at 1 68 Angstroms Resolution J Mol Biol 212 167 184 Kuhn A and Wickner W 1985 Isolation of Mutants in M13 Coat Protein That Affect its Synthesis Processing and Assembly into Phage J Biol Chem 260 15907 15913 LaVallie E R DiBlasio E A Kovacic S Grant K L Schendel P F and McCoy J M 1993 A Thioredoxin Gene Fusion Expression System That Circumvents Inclusion Body Formation in the E coli Cytoplasm Bio Technology 11 187 193 Lee N 1980 Molecular Aspects of ara Regulation In The Operon J H Miller and W S Reznikoff eds Cold Spring Ha
20. CA ATC CTG GTT GAT TTC TGG GCA CAC TGG TGC Ala Ile Leu Val Asp Phe Trp Ala His Trp Cys GAA ATC GCT GAC GAA TAT CAG GGC AAA CTG ACC Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr ACT GCG CCG AAA TAT GGC ATC CGT GGT ATC CCG Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro GCA ACC AAA GTG GGT GCA CTG TCT AAA GGT CAG Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln TCT TTT GAT Ser Phe Asp GGT CCG TGC Gly Pro Cys GTT GCA AAA Val Ala Lys ACT CTG CTG Thr Leu Leu TTG AAA GAG Leu Lys Glu Enterokinase recognition site Enterokinase cleavage site I TCT GGA TCC GGT GAT GAC GAT GAC AAG CTC GCC GAG CGG Ser Gly Ser Gly Asp Asp Asp Asp Lys Leu Ala GGC GAG CTT GAA leer AAG CCT ATC Gly Glu Leu Glu Gly Lys Pro Ile Polyhistidine region Pme I l pBseR I VS epitope CCT AAC CCT CTC CTC GGT CTC GAT TCT acc cer Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg CTT ETT Ac GAMEN TTC Leu Lys m ACC GGT CAT Thr Gly His pBAD Reverse L CAT CAC CAT CAC CAT TGA GTTTAAACG GTCTCCAGCT TGGCTGTTTT GGCGGATGAG AGAAGATTTT CAGCCTGATA His His His His His priming site CAGATTAAAT CAGAACGCAG AAGCGGTCTG ATAAAACAGA ATTTGCCTGG CGGCAGTAGC GCGGTGGTCC CACCTGACCC rrnB T and T transcriptional terminator CATGCCGAAC TCAGAAGTGA AACGCCGTAG CGCCGATGGT AGTGTGGGGT CTCCCCATGC GAGAGTAGGG AACTGCCAGG CATCAAATAA AACGAAAGGC TCAGTCGAAA GACTGGGCCT TTCGTTTTAT CTGTTGTTTG TCGGTGAACG CTCTCCTGAG Producing PCR Products Introduction
21. Invitrogen pBAD TOPO ThioFusion Expression Kit Five minute cloning of Tag polymerase amplified PCR products for soluble regulated expression and purification in E coli Catalog no K370 01 Revision date 21 July 2009 Manual part no 25 0257 MANO000095 ii Contents Kit Contents and Storage nnnnnnsnenensnsenenenenenenrenenenenenenenenenensenenenenenenenenenenenenenenenenseneneneneneneneenenenen iv Introduction nt irereriervennentendendeen ennen end seauscavseseeeneeeaslssav taes soausenseenceunetuedeaeuent 1 D scriptiori ot the System diese edente enti pee Cete ee IHE ER ve Whi Milena 1 Experiment Outline ne Rn een Ret et eeu 3 MEtROOS sintiani akainama ceases keen menkeensoenadrnetenensnrnenandennenndenpnanndsntgenaden 4 Producing PER Products esent a bee deeem eh indt e EH eite i te ene tein teer 6 TOPO Cloning Reaction and Transformation need sti irpo tro i te Pt AREE pae 7 Optimizing the TOPO Cloning Reaction ern edn eru bit rust E HER dbi e RE Qn Feb 12 Expressing the PER Product au tapete maten e dte iaa ded ded 13 Analyzing Samples estan ROG ISBN RR E Re RAD AE ne 16 Purifying Recombinant Prot sta tete nennen nr tenete 18 PDD ONO unten obti de te beta dedifitbeto A dennen 20 RECIPES aes eenoietturseo olini lette unie erem teek cedet eir iios ees 20 Purifying the PCR Products eene eee em nequam aene nd dis 22 Adding 3 A Overhangs Post Amplification ssssssssssssesseenenenenenntet
22. an SDS PAGE gel and electrophorese Save your samples by storing them at 20 C Thaw and resuspend each pellet in 500 uL of Lysis Buffer 2 Freeze sample in dry ice or liquid nitrogen and then thaw at 42 C Repeat 2 to 3 times Note To facilitate lysis you may add lysozyme to the sample or sonicate the cells 3 Centrifuge samples at maximum speed in a microcentrifuge for 1 minute at 4 C to pellet insoluble proteins Transfer supernatant to a fresh tube and store on ice 4 Mixtogether equivalent amounts of supernatant and 2X SDS PAGE sample buffer and boil for 5 minutes 5 Add 500 uL of 1X SDS PAGE sample buffer to the pellets from Step 3 and boil 5 minutes 6 Load 10 uL of the supernatant sample and 5 uL of the pellet sample onto an SDS PAGE gel and electrophorese To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels and electrophoresis apparatus 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 refer to www invitrogen com or contact T
23. atin dNTP Mix 12 5 mM dATP 10 uL 12 5 mM dCTP 12 5 mM dGTP 12 5 mM dTTP neutralized at pH 8 0 in water Salt Solution 1 2 M NaCl 50 uL 0 06 M MgCl 20 L Arabinose 20 in sterile water 1 mL Trx Forward 0 1 pg pL in TE Buffer pH 8 20 pL Sequencing Primer pBAD Reverse 0 1 ug uL in TE Buffer pH 8 20 pL Sequencing Primer Control PCR Primers 0 1 pg pL in TE Buffer pH 8 10 uL Control PCR 0 05 ug uL in TE Buffer pH 8 10 uL Template Continued on next page Kit Contents and Storage Continued pBAD Thio TOPO TA Cloning Reagents continued Sequences of the Primers One Shot Reagents Genotype of TOP10 Item Concentration Amount Sterile Water 1 mL Expression Control Plasmid 500 ng uL in TE buffer 10 pL pBAD Thio supercoiled pH8 The table below provides the sequences of the Trx Forward and pBAD Reverse sequencing primers Two micrograms of each primer are supplied Primer Sequence pMoles Supplied Trx Forward 5 TITCCICGACGCTAACCTG 3 371 pBAD Reverse 5 GATITAATCTGTATCAGG 3 363 The table below describes the items included in the One Shot TOP10 Chemically Competent E coli kit Store at 80 C Item Composition Amount TOP10 cells 21 x 50 uL SOC Medium 2 Tryptone 6 mL may be stored atroom 0 5 Yeast Extract temperature or 4 C 10 mM NaCl 25mMKCI 10 mM MgCl 10 mM MgSO4 20 mM glucose pUC19 Control DNA 10 pg uL in 5 mM Tris 50
24. covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple
25. duct Features pBAD TOPO ThioFusion Expression Kit provides a highly efficient 5 minute one step cloning strategy TOPO Cloning for the direct insertion of Taq polymerase amplified PCR products into a plasmid vector for soluble regulated expression and simplified protein purification in F coli No ligase post PCR procedures or PCR primers containing specific sequences are required Expression in E coli is driven by the araBAD promoter Psap The AraC gene product encoded on the pBAD Thio TOPO plasmid positively regulates this promoter Recombinant proteins are expressed as fusions to His Patch thioredoxin for high level expression and simple purification TOPO Cloning The PCR expression vector pBAD Thio TOPO is supplied linearized with e Single3 thymidine T overhangs for TA Cloning e Topoisomerase I bound to the vector this is referred to as activated vector Tag polymerase has a nontemplate dependent terminal transferase activity that adds a single deoxyadenosine A to the 3 ends of PCR products The linearized vector supplied in this kit has single overhanging 3 deoxythymidine T residues This allows PCR inserts to ligate efficiently with the vector Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5 CCCTT in one strand Shuman 1991 The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between
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27. e 11 7 kDa thioredoxin protein is found in yeast plants and mammals as well as in bacteria It was originally isolated from E coli as a hydrogen donor for ribonuclease reductase for a review see Holmgren 1985 The gene has been completely sequenced Wallace and Kushner 1984 The protein has been crystallized and its three dimensional structure determined Katti et al 1990 When overexpressed in E coli thioredoxin is able to accumulate to approximately 4076 of the total cellular protein and still remains soluble Thioredoxin is used to increase translation efficiency and in some cases solubility of eukaryotic proteins expressed in E coli Murine interleukin 2 human interleukin 3 murine interleukin 4 murine interleukin 5 human macrophage colony stimulating factor murine steel factor murine leukemia inhibitory factor and human bone morphogenetic protein 2 are some of the proteins that have been produced as soluble C terminal fusions to the thioredoxin protein in E coli LaVallie et al 1993 To create a metal binding domain in the thioredoxin protein the glutamate residue at position 32 and the glutamine residue at position 64 were mutated to create histidine residues When His Patch thioredoxin folds the histidines at positions 32 and 64 interact with a native histidine at position 8 to form a patch This histidine patch was shown to have high affinity for divalent cations Lu et al 1996 His Patch thioredoxin HP thio
28. e tenete 24 PBAD TOPO ThioFusion Control React ONS cuc p erred PLA Dus ibd eneen 25 Troubleshooting a eee edt tette HE EH ceret ate rola te id rese eb re takes 27 Map and Features of pDADZVIUo TOP Batenstein 28 Map otpBAD Thio REET EA A E 30 Regulation by ArabihosE tsetera osteen iaai seque endorse ebd ne dedii peas 31 Accessory Products cios eee ene tee ent amati E 32 Technical Support 34 5 trien inan E GUI ERI E A E EE UI RERUM ERE NR 34 Purchaser Notifications sigg a E a e a a E E E a e a e A O ETER 35 References innam then ata N E a E eS 37 iii Kit Contents and Storage Shipping and Storage pBAD Thio TOPO TA Cloning Reagents TM The pBAD TOPO ThioFusion Expression Kit is shipped on dry ice Each kit contains a box with pBAD Thio TOPO TA Cloning reagents Box 1 a box with One Shot TOP10 Chemically Competent E coli Box 2 and a stab of LMG194 Store Box 1 at 20 C and Box 2 at 80 C Store the LMG194 stab at 4 C pBAD Thio TOPO TA Cloning reagents Box 1 are listed below Note that you must supply the Taq polymerase Store Box 1 at 20 C Item Concentration Amount pBAD Thio TOPO 10 ng uL plasmid DNA in 20 reactions vector linearized 50 glycerol 50 mM Tris HCI pH 7 4 at 25 C 1 mM EDTA 2 mM DTT 0 1 Triton X 100 100 ug mL BSA 30 uM phenol red 10X PCR Buffer 100 mM Tris HCI pH 8 3 at 42 C 100 uL 500 mM KCI 25 mM MgCl 0 0176 gel
29. echnical Support page 34 Continued on next page Analyzing Samples Continued Analyzing Samples Low Expression Optimizing Expression Stain the gel and look for a band of increasing intensity in the expected size range for the recombinant protein Use the positive control pBAD Thio to confirm that growth and induction was done properly The positive control should yield a 16 kDa protein when induced with 0 02 arabinose Determine the approximate arabinose concentration for maximum expression If you don t see any expression on a gel re run your samples on an SDS PAGE gel and perform a western blot Use antibody to your protein or any of the antibodies listed on page 33 If you still don t see expression of your protein sequence your construct and make sure it is in frame with the N terminal and or C terminal peptide After you have detected expression of your protein of interest perform some experiments to further optimize expression Use the Pilot Expression protocol on pages 14 15 but vary the arabinose concentration over a smaller range For example if you obtained the best expression at 0 002 arabinose in the medium try 0 0004 0 0008 0 001 0 004 and 0 008 You may also perform a time course of induction to determine if varying the time increases expression Take time points every hour over a5 to 6 hour period If your protein is insoluble analyze the supernatant and pellet of lysed c
30. ells when you vary the arabinose concentration see Preparing Samples for Soluble Insoluble Protein previous page Store your cell lysates at 20 C 17 Purifying Recombinant Protein Introduction ProBond Note Additional Purification Steps Scaling Up Expression for Purification on ProBond 18 After you have expressed your recombinant fusion protein you are ready to purify your fusion protein using a metal chelating resin such as ProBond ProBond is a nickel charged Sepharose resin that can be used for affinity purification of fusion proteins containing the HP thioredoxin leader peptide and or a 6xHis tag Proteins bound to the resin may be eluted with either low pH buffer or competition with imidazole or histidine e To scale up your pilot expression for purification see below TM e To purify your fusion protein using ProBond refer to the ProBond Purification manual e To purify your fusion protein using another metal chelating resin refer to the manufacturer s instructions Note that denaturing conditions will destroy the Ni binding site created by the histidine patch in HP thioredoxin There may be cases when your specific HP thioredoxin fusion protein may not be completely purified by metal affinity chromatography Other protein purification techniques may be utilized in conjunction with ProBond to purify the fusion protein Deutscher 1990 The capacity of Pr
31. emethylation Reactions J Bacteriol 171 3609 3618 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press San Millan J L Boyd D Dalbey R Wickner W and Beckwith J 1989 Use of phoA Fusions to Study the Topology of the Escherichia coli Inner Membrane Protein Leader Peptidase J Bacteriol 171 5536 5541 Schleif R S 1992 DNA Looping Ann Rev Biochem 61 199 223 Shuman S 1994 Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase J Biol Chem 269 32678 32684 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Wallace B J and Kushner S R 1984 Genetic and Physical Analysis of the Thioredoxin trxA Gene of Escherichia coli K 12 Gene 32 399 408 2009 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use Sepharose is a registered trademark of Amersham Pharmacia Biotech Vent is a registered trademark of New England BioLabs 38 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA
32. formant and two samples for each control To ensure low levels of expression you may find it useful to utilize 0 2 glucose to repress the araBAD promoter further Follow the steps below to express your protein 1 Transform your construct into LMG194 LMG194 can be grown in RM medium that enables repression of Pgap by glucose or glycerol Follow the Pilot Expression protocol using RM medium containing 0 2 glycerol to grow the cells i e substitute glycerol for glucose in the media recipe on page 21 Be sure to monitor the ODo as the cells will grow more slowly in RM medium Induce with various concentrations of arabinose as described in the Pilot Expression Monitor ODe over time to be sure cells are growing 15 Analyzing Samples Materials Needed Preparing Samples Preparing Samples for Soluble Insoluble Protein Polyacrylamide Gel Electrophoresis 16 e Reagents and apparatus for SDS PAGE gel e 1Xand 2X SDS PAGE sample buffer e Boiling water bath e Lysis Buffer see page 21 for recipe e Liquid nitrogen optional Before starting prepare SDS PAGE gels or use one of the pre cast polyacrylamide gels available from Invitrogen see next page to analyze the collected samples 1 When all the samples have been collected from the pilot expression resuspend each cell pellet in 100 uL of 1X SDS PAGE sample buffer Boil 5 minutes and centrifuge briefly Load 5 10 uL of each sample on
33. fter purification add Taq polymerase buffer dATP and 0 5 unit of Tag polymerase and incubate 10 15 minutes at 72 C Use 4 uL in the TOPO Cloning reaction pBAD TOPO ThioFusion Control Reactions Introduction Before Starting Producing Control PCR Product If you have trouble obtaining transformants or vector containing insert perform the following control reactions to help troubleshoot your experiment Performing the control reactions involves producing a control PCR product containing the lac promoter and the LacZa fragment using the reagents included in the kit Successful TOPO Cloning of the control PCR product will yield blue colonies on LB agar plates containing antibiotic and X gal Prepare the following reagents before performing the control reaction e 40 mg mL X gal in dimethylformamide e LB plates containing 50 100 pg mL ampicillin and X gal see page 20 1 To produce the 500 bp control PCR product containing the lac promoter and LacZa set up the following 50 uL PCR Control DNA Template 50 ng 1uL 10X PCR Buffer 5 pL 50 mM dNTPs 0 5 uL Control PCR Primers 0 1 ug uL 1 pL Sterile Water 41 5 pL Tag Polymerase 1 unit uL lul Total Volume 50 pL 2 Amplify using the following cycling parameters Step Time Temperature Cycles Initial Denaturation 2 minutes 94 C 1X Denaturation 1 minute 94 C Annealing 1 minute 55 C 25X Extension 1 minute 72 C Final Extension 7 minutes 72
34. g Procedure Note 24 Direct cloning of DNA amplified by Vent or Pfu polymerases into TOPO Cloning vectors is often difficult because of very low cloning efficiencies These low efficiencies are caused by the lack of the terminal transferase activity that adds the 3 A overhangs necessary for TOPO Cloning Invitrogen has developed a simple method to clone these blunt ended fragments Tag polymerase e A heat block equilibrated to 72 C e Phenol chloroform optional e 3M sodium acetate optional e 100 ethanol optional e 80 ethanol optional e TE buffer optional This is just one method for adding 3 adenines Other protocols are also suitable 1 Afteramplification with Vent or Pfu polymerase place vials on ice and add 0 7 1 unit of Tag polymerase per tube Mix well It is not necessary to change the buffer Incubate at 72 C for 8 10 minutes do not cycle Place the vials on ice The DNA amplification product is now ready for ligation into pBAD Thio TOPO Note If you plan to store your sample s overnight before proceeding with TOPO Cloning you may want to extract your sample s with phenol chloroform to remove the polymerases After phenol chloroform extraction precipitate the DNA with ethanol and resuspend the DNA in TE buffer to the starting volume of the amplification reaction You may also gel purify your PCR product after amplification with Vent or Pfu see previous page for protocol A
35. he amount of surrounding agarose excised with the fragment Weigh the gel slice Add Gel Solubilization Buffer GS1 supplied in the kit as follows e For lt 2 agarose gels place up to 400 mg gel into a sterile 1 5 mL polypropylene tube Divide gel slices exceeding 400 mg among additional tubes Add 30 uL Gel Solubilization Buffer GS1 for every 10 mg of gel e For gt 2 agarose gels use sterile 5 mL polypropylene tubes and add 60 pL Gel Solubilization Buffer GS1 for every 10 mg of gel Incubate the tube at 50 C for 15 minutes Mix every 3 minutes to ensure gel dissolution After gel slice appears dissolved incubate for an additional 5 minutes Preheat an aliquot of TE Buffer TE to 65 70 C Place a Quick Gel Extraction Column into a Wash Tube Pipette the mixture from Step 4 above onto the column Use 1 column per 400 mg agarose Centrifuge at gt 12 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube Optional Add 500 uL Gel Solubilization Buffer GS1 to the column Incubate at room temperature for 1 minute Centrifuge at gt 12 000 x g for 1 minute Discard the flow through Place the column back into the Wash Tube Add 700 uL Wash Buffer W9 with ethanol add 96 100 ethanol to the Wash Buffer according to instructions on the label of the bottle to the column and incubate at room temperature for 5 minutes Centrifuge at 212 000 x g for 1 minute Discard flow through Centrifuge the c
36. ing Invitrogen s ProBond Resin see below To purify proteins expressed using pBAD Thio TOPO the ProBond Purification System is available separately Additional ProBond resin is available in bulk See the table below for ordering information Product Amount Cat no 6 purifications K850 01 TM ProBond Purification System includes six 2 mL precharged prepacked ProBond resin columns and buffers for native and denaturing purification ProBond Purification Kit with Anti V5 HRP 1 kit K854 01 Antibody ProBond Metal Binding Resin 50 mL R801 01 precharged resin provided as a 50 slurry in 20 ethanol 150 mL R801 15 Purification Columns 50 columns R640 50 10 mL polypropylene columns Note that under denaturing conditions the Ni binding site encoded by the histidine patch will be destroyed because the HP thioredoxin protein will be denatured The binding of nickel ion to the 6xHis tag is not affected by denaturing conditions 33 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs 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 assistanc
37. ion from Step 2 previous page into a vial of One Shot Chemically Competent E coli and mix gently Do not mix by pipetting up and down Incubate on ice for 5 to 30 minutes Note Longer incubations on ice do not seem to have any affect on transformation efficiency The length of the incubation is at the user s discretion see page 12 Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 250 uL of room temperature SOC medium Cap the tube tightly and shake horizontally 200 rpm at 37 C for 1 hour Spread 25 200 uL from each transformation on a prewarmed selective plate and incubate overnight at 37 C We recommend that you plate two different volumes to ensure that at least one plate will have well spaced colonies An efficient TOPO Cloning reaction will produce hundreds of colonies Pick 10 colonies for analysis see Analyzing Positive Clones next page Add 2 uL of the TOPO Cloning reaction into a 0 1 cm cuvette containing 50 ul of electrocompetent E coli and mix gently Do not mix by pipetting up and down Avoid formation of bubbles Electroporate your samples using the protocol for your electroporator Note If you have problems with arcing see below Immediately add 250 uL of room temperature SOC medium Transfer the solution to a 15 mL snap cap tube and shake for at least 1 hour at 37 C to allow expression of the antibiotic resistance gene Spread 10 50 uL from each
38. ion is diluted 4 fold to prepare a 300 mM NaCl 15 mM MgCl solution for convenient addition to the TOPO Cloning reaction see next page e 42 C water bath or electroporator with cuvettes optional e LB plates containing 50 100 pg mL ampicillin two for each transformation e 37 C shaking and non shaking incubator Continued on next page TOPO Cloning Reaction and Transformation Continued Preparing for Transformation Setting Up the TOPO Cloning Reaction Performing the TOPO Cloning Reaction For each transformation you need one vial of competent cells and two selective plates e Equilibrate a water bath to 42 C for chemical transformation or set up your electroporator if you are using electrocompetent E coli e For electroporation dilute a small portion of the Salt Solution 4 fold to prepare Dilute Salt Solution e g add 5 uL of the Salt Solution to 15 uL of sterile water e Warm the vial of SOC medium from Box 2 to room temperature e Warm selective plates at 37 C for 30 minutes e For each transformation thaw 1 vial of One Shot cells on ice The table below describes how to set up your TOPO Cloning reaction 6 uL for eventual transformation into chemically competent TOP10 One Shot E coli provided or electrocompetent E coli Refer to page 11 for additional information on optimizing the TOPO Cloning reaction An Insert vector molar ratio of 1 1 gives the optimal efficiency in TOPO C
39. limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 or outlicensing lifetech com 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 Continued on next page 35 Purchaser Notification Continued Limited Use Label The ThioFusion
40. loning reaction Note The red color of the TOPO vector solution is normal and is used to visualize the solution Reagent Chemically Competent Electrocompetent E coli E coli Fresh PCR product 0 5 to 4 uL 0 5 to 4 uL Salt Solution 1 uL Dilute Salt Solution 1 uL Sterile Water add to a total volume of 5 add to a total volume of 5 uL uL TOPO vector 1 pL 1 pL Final Volume 6 pL 6 pL Store all reagents at 20 C when finished Salt solutions and water can be stored at room temperature or 4 C 1 Mix reaction gently and incubate for 5 minutes at room temperature Note For most applications incubation for 5 minutes yields plenty of colonies for analysis Depending on your needs you can vary the length of the TOPO Cloning reaction from 30 seconds to 30 minutes For routine subcloning of PCR products 30 seconds is sufficient For large PCR products 1 kb or if you are TOPO Cloning a pool of PCR products increasing the reaction time yields more colonies 2 Place the reaction on ice and proceed to the One Shot Chemical Transformation or Transformation by Electroporation next page Note You may store the TOPO Cloning reaction at 20 C overnight Continued on next page TOPO Cloning Reaction and Transformation Continued One Shot TOP10 Chemical Transformation Transformation by Electroporation Note 1 n le 10 Add 2 pL of the TOPO Cloning react
41. nus of the protein see page 5 To digest your fusion protein with enterokinase follow the manufacturer s recommendations A recombinant preparation of the catalytic subunit of bovine enterokinase EKMax is available from Invitrogen Instructions for digestion are included with the product To remove EKMax from the digest you may use EK Away Resin also available from Invitrogen see page 32 for ordering information 19 Recipes LB Luria Bertani Medium and LB Agar Plates X Gal Stock Solution SOB Medium with Ampicillin 20 Appendix 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 Forlliter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 mL deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and volume to 1 liter 3 Autoclave on liquid cycle for 20 minutes at 15 psi Allow solution to cool to 55 C and add antibiotic if needed 50 100 ug mL ampicillin 4 Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes at 15 psi 3 After autoclaving cool to 55 C add antibiotic 50 100 pg mL of ampicillin and pour into 10 cm plates 4 Letharden then invert and store at 4 C in the dark 5 To add X gal to the plate warm the plate to 37 C Pipette 40 uL of the 40 mg mL stock solution see below onto the plate spread evenly and let dry 1
42. oBond is about 1 mg of protein per mL Depending on the expression level of your recombinant fusion protein you may need to adjust the culture volume to bind the maximum amount of recombinant fusion protein For a TM prepacked 2 mL ProBond column start with 50 mL of bacterial culture To grow and induce a 50 mL bacterial culture 1 Inoculate 2 mL of SOB or LB containing 50 100 ug mL ampicillin with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 250 rpm to ODeoo 1 2 The next day inoculate 50 mL of SOB or LB containing 50 100 pg mL ampicillin with 1 mL of the overnight culture 4 Grow the culture at 37 C with vigorous shaking to an OD 0 5 the cells should be in mid log phase Add the optimal amount of arabinose to induce expression Grow at 37 C with shaking until the optimal time point is reached Harvest the cells by centrifugation 3 000 x g for 10 minutes at 4 C 7 Atthis point you may proceed directly to purification ProBond Purification System manual or store the cells at 80 C for future use Continued on next page Purifying Recombinant Protein Continued Removing the N terminal Leader The enterokinase recognition site in the HP thioredoxin leader may be utilized to remove the leader sequence from your protein after purification Note that after digestion with enterokinase there will be three vector encoded amino acids Leu Ala Leu remaining at the N termi
43. olumn at 212 000 x g for 1 minute to remove any residual buffer Place the column into a 1 5 mL Recovery Tube Add 50 uL warm 65 70 C TE Buffer TE to the center of the cartridge Incubate at room temperature for 1 minute Centrifuge at gt 12 000 x g for 2 minutes The Recovery Tube contains the purified DNA Store DNA at 20 C Discard the column Use 4 pL of the purified DNA for the TOPO Cloning reaction Continued on next page Purifying the PCR Products Continued Low Melt Agarose Note that gel purification will dilute your PCR product Use only chemically Method competent cells for transformation 1 Electrophorese as much as possible of your PCR reaction on a low melt agarose gel 0 8 to 1 270 in TAE buffer Visualize the band of interest and excise the band Place the gel slice in a microcentrifuge tube and incubate the tube at 65 C until the gel slice melts Place the tube at 37 C to keep the agarose melted Use 4 uL of the melted agarose containing your PCR product in the TOPO Cloning reaction page 7 Incubate the TOPO Cloning reaction at 37 C for 5 to 10 minutes This is to keep the agarose melted Transform 2 to 4 uL directly into TOP10 One Shot cells using the method on page 9 Note Cloning efficiency may decrease with purification of the PCR product To produce a single band optimize your PCR conditions 23 Adding 3 A Overhangs Post Amplification Introduction Before Startin
44. pport see page 34 Nco Pme HP thioredoxin EK site 1 V5 epitope 6xHis BStop Comments for pBAD Thio 4436 nucleotides Arabinose O operator region bases 4 19 Arabinose O4 operator region bases 161 182 CAP binding site bases 203 216 Arabinose I4 and l2 region bases 213 251 Arabinose minimal promoter bases 248 276 Ribosome binding site bases 329 332 His Patch Thioredoxin ORF bases 346 674 Trx Forward priming site bases 655 672 Enterokinase recognition site bases 691 705 V5 epitope bases 712 753 Polyhistidine region bases 763 783 pBAD Reverse priming site bases 836 853 rrnB transcriptional termination region bases 886 1043 Ampicillin resistance gene ORF bases 1323 2183 pUC origin bases 2328 3001 AraC ORF bases 3532 4410 opposite strand Regulation by Arabinose Regulation of the Pgap Promoter Glucose Repression The araBAD promoter used in pBAD Thio TOPO is both positively and negatively regulated by the product of the araC gene Ogden et al 1980 Schleif 1992 AraC is a transcriptional regulator that forms a complex with L arabinose In the absence of arabinose the AraC dimer contacts the O and I half sites of the araBAD operon forming a 210 bp DNA loop see the figure below For maximum transcriptional activation two events are required Arabinose binds to AraC and causes the protein to release the O site and bind the I site which is adjacent to the I site This release
45. rbor N Y Cold Spring Harbor Laboratory pp 389 410 Lee N Francklyn C and Hamilton E P 1987 Arabinose Induced Binding of AraC Protein to aral Activates the araBAD Operon Promoter Proc Natl Acad Sci USA 84 8814 8818 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 Lu Z DiBlasio Smith E A Grant K L Warne N W LaVallie E R Collins Racie L A Follettie M T Williamson M J and McCoy J M 1996 Histidine Patch Thioredoxins J Biol Chem 271 5059 5065 Continued on next page 37 References Continued Miyada C G Stoltzfus L and Wilcox G 1984 Regulation of the araC Gene of Escherichia coli Catabolite Repression Autoregulation and Effect on araBAD Expression Proc Natl Acad Sci USA 81 4120 4124 Ogden S Haggerty D Stoner C M Kolodrubetz D and Schleif R 1980 The Escherichia coli L Arabinose Operon Binding Sites of the Regulatory Proteins and a Mechanism of Positive and Negative Regulation Proc Natl Acad Sci USA 77 3346 3350 Russell C B Stewart R C and Dahlquist F W 1989 Control of Transducer Methylation Levels in Escherichia coli Investigation of Components Essential for Modulation of Methylation and D
46. redoxin Provides a highly efficient fusion partner for translation of the fusion protein TOPO Cloning site Allows quick insertion of your PCR product for expression C terminal V5 epitope tag Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of the fusion protein by the Anti V5 Antibody or the Anti V5 HRP Antibody Southern et al 1991 C terminal polyhistidine region optional Permits purification of recombinant fusion protein on metal chelating resins i e ProBond In addition it allows detection of the recombinant protein with the Anti His C term Antibody or the Anti His C term HRP Antibody Lindner et al 1997 rrnB transcription termination region Strong transcription termination region Ampicillin resistance gene f lactamase Allows selection of the plasmid in F coli pUC origin Replication and growth in E coli araC gene Encodes the regulatory protein for tight regulation of the Pgap promoter Lee 1980 Schleif 1992 29 Map of pBAD Thio Description pBAD Thio is a 4 436 bp control vector expressing a 16 kDa HP thioredoxin fusion protein The whole fusion gene may be excised with Nco I and Pme I Map of The figure below summarizes the features of the pBAD Thio vector The complete Control Vector nucleotide sequence for pBAD Thio is available for downloading at 30 www invitrogen com or by contacting Technical Su
47. redoxin proteins can therefore be purified on metal chelating resins e g ProBond See page 33 for ordering information Experiment Outline Experiment Flowchart The flow chart below describes the general steps needed to amplity TOPO Clone and express your protein of interest Design Primers for PCR Produce PCR product TOPO Cloning Reaction Mix together PCR product and pBAD Thio TOPO vector at room temperature Transform into TOP10 E coli cells Incubate 5 minutes lt select and analyze colonies gt Select and analyze colonies Select a positive transformant and induce expression with arabinose Methods Designing PCR Primers Introduction It is important to properly design your PCR primers to ensure that you obtain the recombinant protein you need for your studies Use the information below and the diagram on the next page to design your PCR primers Considerations pBAD Thio TOPO is designed with some specific features to facilitate expression They are e The initiation ATG is correctly spaced from the optimized ribosome binding site to ensure optimal translation e HP thioredoxin acts as a translation leader to facilitate high level expression and in some cases solubility Note You can remove HP thioredoxin after protein purification using enterokinase i e EXMax see page 19 Primer Design Suggestions for primer design are provided in the table below
48. rotein pBAD Thio is included for use as a positive expression control TOP10 cells may be used as a general host for expression The E coli strain LMG194 Guzman et al 1995 is included in the kit to allow additional repression for low basal level expression of toxic genes This strain is capable of growth on minimal medium RM medium which allows repression Of Psap by glucose After you have determined that you have the correct construct transform it into LMG194 prior to performing expression experiments Follow the guidelines below for using LMG194 e Induce the pBAD promoter when cells are growing in LB or RM Glucose e If you are growing your construct under maximal repression i e with D glucose in RM media then you must spin down the culture and resuspend it in RM containing 0 2 glycerol and Arabinose i e substitute glycerol for the glucose in the media recipe on page 21 The positive control vector pBAD Thio is included in the kit as an expression control Details of this vector are provided on page 28 Transform the vector 10 ng into TOP10 cells using the procedure on page 9 Once you have some clones that you wish to characterize we recommend the following strategy to determine the optimal expression level 1 Pilot Expression Vary the amount of arabinose over a 10 000 fold range 0 00002 to 0 2 to determine the approximate amount of arabinose needed for maximum expression of your protein See next page
49. rtifacts false positives TOPO Cloning is very efficient for small fragments 100 bp present in certain PCR reactions Gel purify your PCR product page 22 or optimize your PCR If your template DNA carries an ampicillin marker carryover into the TOPO Cloning reaction from the PCR may lead to false positives Linearize the template DNA prior to PCR to eliminate carryover PCR product does not contain sufficient 3 A overhangs even though you used Tag polymerase Taq polymerase is less efficient at adding a nontemplate 3 A next to another A Taq is most efficient at adding a nontemplate 3 A next to a C You may have to redesign your primers so that they contain a 5 G instead of a 5 T Brownstein et al 1996 27 Map and Features of pBAD Thio TOPO pBAD Thio TOPO The map below shows the features of pBAD Thio TOPO The complete Map sequence of the vector is available for downloading at www invitrogen com or from Technical Support page 34 PCR Product A A vs epitope FoxHis fStop Nco I Pme I pBAD Thio TOPO Comments for pBAD Thio TOPO 4454 nucleotides Arabinose O2 operator region bases 4 19 Arabinose O1 operator region bases 161 182 CAP binding site bases 203 216 Arabinose l1 and 12 region bases 213 251 Arabinose minimal promoter bases 248 276 Ribosome binding site bases 329 334 His Patch Thioredoxin ORF bases 346 674 Trx Forward priming site bases 655
50. s the DNA loop and allows transcription to begin The cAMP activator protein CAP CAMP complex binds to the DNA and stimulates binding of AraC to I and L AraC dimer No transcription arabinose Transcription Basal expression levels can be repressed by introducing glucose to the growth medium Glucose acts by lowering cAMP levels which in turn decreases the binding of CAP As cAMP levels are lowered transcriptional activation is decreased 31 Accessory Products Additional Many of the reagents supplied with the pBAD TOPO ThioFusion Expression Products Kit and other reagents suitable for use with the kit are available separately from Invitrogen Ordering information for these reagents is provided below For details visit www invitrogen com Product Amount Cat no Platinum Tag DNA Polymerase 100 reactions 10966 018 250 reactions 10966 026 500 reactions 10966 034 Taq DNA Polymerase Recombinant 100 units 10342 053 500 units 10342 020 PCR Optimizer Kit 100 reactions K1220 01 PCR SuperMix High Fidelity 100 reactions 10790 020 One Shot TOP10 Chemically Competent 10 reactions C4040 10 coe 20 reactions C4040 03 PureLink HO Mini Plasmid Purification Kit 100 preps K2100 01 PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 100 preps K2100 03 EKMax Enterokinase 250 units E180 01 EK Away Resin 7 5mL R180 01 Ampicillin Sodium Salt
51. te and these should be all white pUC19 plasmid is included as a control to check the transformation efficiency of One Shot competent cells Transform One Shot competent cells with 10 pg of plasmid per 50 uL of cells using the protocol on page 10 Plate 10 uL of the transformation mixture plus 20 uL of SOC to help ensure even spreading on LB plates containing 100 ug mL ampicillin Transformation efficiency should be 1x 10 cfu ug DNA Continued on next page Troubleshooting Factors Affecting Lower cloning efficiencies may be a result from different variables Most of these Cloning Efficiency are easily correctable but if you are cloning large inserts you may not obtain the expected 90 or more cloning efficiency Variable Solution pH 9 in PCR amplification reaction Check the pH of the PCR amplification reaction and adjust with 1 M Tris HCI pH 8 Incomplete extension during PCR Be sure to include a final extension step of 7 to 30 minutes during PCR Longer PCR products will need a longer extension time Cloning large inserts gt 3 kb Increase amount of insert or gel purify as described on page 22 Excess or overly dilute PCR product Reduce or concentrate the amount of PCR product You may add up to 4 pL of your PCR to the TOPO Cloning reaction page 8 Cloning blunt ended fragments Add 3 A overhangs by incubating with Taq polymerase page 24 PCR cloning a
52. uL HCI 0 5 mM EDTA pH 8 0 TOP10 Use this strain for general cloning and expression of PCR products in pBAD Thio TOPO This strain cannot be used for single strand rescue of DNA F mcr A A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 recA1 araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG Continued on next page Kit Contents and Storage Continued Genotype of F AlacX74 gal E thi rpsL AphoA Pou II Aara714 leu Tn10 LMG194 Note This strain is deleted for araBADC and is streptomycin and tetracycline resistant Storing LMG194 The LMG194 E coli cells supplied as a stab with the kit are guaranteed until the Stab expiration date marked on tube when stored at 4 C Upon receipt we recommend that you prepare a set of glycerol master stocks within two weeks of receiving the kit To prepare 5 10 glycerol master stocks for long term storage 1 Streak a small portion of the LMG194 cells that you have received as a stab on an LB plate containing the appropriate antibiotics Invert the plate and incubate at 37 C overnight 3 Isolate a single colony and inoculate into 5 10 mL of LB medium containing the appropriate antibiotics 4 Grow the culture to stationary phase OD 1 2 5 Mix0 8 mL of culture with 0 2 mL of sterile glycerol and transfer to a cryovial Store at 80 C Use one master stock to create working stocks for regular use vi Introduction Description of the System Pro
53. ure Plasmid Miniprep kits see page 32 for ordering information Refer to www invitrogen com or contact Technical Support for more information on a large selection of plasmid purification columns Because the PCR product will clone in either direction analyze for orientation as well as insertion by restriction analysis or by sequencing The Trx Forward and pBAD Reverse sequencing primers are included to sequence your insert Refer to the diagram on page 5 for primer binding sites Alternative You may directly analyze positive transformants using PCR You may use the Trx Method of Forward and pBAD Reverse sequencing primers as PCR primers We recommend Analysis performing restriction analysis in parallel to confirm that PCR gives you the correct result Artifacts can be obtained because of mispriming or contaminating template The following protocol is provided for your convenience Other protocols are suitable 1 Prepare a PCR cocktail consisting of PCR buffer dNTPs primers and Taq polymerase Use a 20 uL reaction volume Multiply by the number of colonies to be analyzed e g 10 2 Pick 10 colonies and resuspend them individually in 20 uL of the PCR cocktail 3 Incubate the reaction for 10 minutes at 94 C to lyse the cells and inactivate nucleases 4 Amplify for 20 to 30 cycles 94 C for 1 minute 55 C for 1 minute and 72 C for 1 minute 5 For the final extension incubate at 72 C for 10 minutes Hold at 4 C
54. y 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 warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please 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 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Use of the pBAD TOPO ThioFusion Expression Kit is
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