pBAD/His A, B, and C pBAD/Myc
Contents
1. 02 Region I 1 AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT 61 TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA O4 Region I 121 AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward priming site I 181 ATTATTTGCA CGGCGTCACA CTTTGCTATG sn TTTATCCATA AGATTAGCGG 12 and l4 Region 35 10 241 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TITITTGGGC EAS RBS Nco Polyhistidine Region 301 TAACAGGAGG AATTAACC ATG GGG GGT TCT car CAT CAT CAT CAT CAT GGT ATG GCT Met Gly Gly Ser His His His His His His Gly Met Ala EK recognition site Xpress Epitope 358 AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys EK cleavage site A Xhol Ba Il Pst Kpn EcoR I I I I I 412 GAT CGA TGG ATC CGA CCT CGA GAT CTG CAG ATG GTA CCA TAT GGG AAT Asp Arg Trp Ile Arg Pro Arg Asp Leu Gln Met Val Pro Tyr Gly Asn Sful Hind Ill pBAD reverse priming site I 460 TCG AAG CTT GGCTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC Ser Lys Leu 519 AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC rrnB T4 and Ta transcriptional terminator 579 ACCTGACCCC ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC 639 TCCCCATGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ape2. 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 Pgap Promoter J Bacteriol 177 4121 4130 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 Lee N 1980 Molecular Aspects of ara Regulation In The Operon J H Miller and W S Reznikoff eds Cold Spring Harbor 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 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 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 Demethylation 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 Ha
3. A 699 ACTGGGCCTT TCGTTTTATCT 11 Cloning into pBAD Myc His Important Note pBAD Myc His Multiple Cloning Sites To generate recombinant proteins that are expressed correctly and contain the C terminal fusion peptide it is necessary to clone in frame with BOTH the initiation ATG bp 320 322 and the C terminal peptide The initiation ATG is correctly spaced from the optimized RBS to ensure optimum translation To facilitate cloning the pBAD Myc His vector is provided in three different reading frames They differ only in the spacing between the sequences that code for the multiple cloning site and the C terminal peptide For proper expression first determine which restriction sites are appropriate for ligation and then which vector will preserve the reading frame at BOTH the 5 and the 3 ends You may have to use PCR to create a fragment with the appropriate restriction sites to clone in frame at both ends Be sure that there is no stop codon in the open reading frame of your gene except as noted below If you wish to express your protein WITHOUT the C terminal peptide be sure to include a stop codon at the end of your protein The multiple cloning sites of each version of pBAD Myc His are provided on pages 13 15 Restriction sites are labeled to indicate cleavage site The boxed sequence is the variable region that facilitates in frame cloning with the ATG codon and C terminal peptide This variable region is loc
4. O4 Region I 121 AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward priming site 181 ATTATTTGCA CGGCGTCACA CTTIGCTATG Sn TTTATCCATA AGATTAGCGG 12 and l4 Region 35 10 241 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TITTTTGGGC a RBS Nco Xhol Sac Bg Il Pst Kpn l Lt l 301 TAACAGGAGG AATTAACC ATG GATCCGAGCT CGAGATCTGC AGCTGGTACC ATATG Met EcoRI Sful Hind Ill myc epitope I I I 357 GGAATTCGAA GCTTGGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Polyhistidine Region I 413 GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTAAACGG TCTCCAGCTT GGCTGTTTTG Ala Val Asp His His His His His His pBAD reverse priming site 473 GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA 533 TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT rrnB T4 and Ta transcriptional terminators 593 CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA 653 ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC Continued on next page 13 Cloning into pBAD Myc His continued pBAD Myc His B Below is the multiple cloning site for pBAD Myc His B Restriction sites are Multiple Cloning labeled to indicate the cleavage site The boxed nucleotides indicate the variable Site 61 121 181 241 3
5. Provides a translational initiation site for the fusion protein N terminal polyhistidine tag Forms metal binding site for affinity purification of recombinant fusion protein on a metal chelating resin In addition it allows detection of the recombinant protein with the Penta His Mouse IgG1 Monoclonal Antibody see page vi for ordering information Anti Xpress epitope Permits detection of recombinant Asp Leu Tyr Asp Asp Asp Asp Lys fusion protein by appropriate antibodies see page vi for ordering information Enterokinase cleavage site Allows removal of the N terminal Asp Asp Asp Asp Lys peptide by enterokinase for production of native protein see page vi for ordering information Multiple cloning site Allows insertion of your gene for expression rrnB transcription termination region Strong transcription termination region Ampicillin resistance gene Allows selection of the plasmid in P lactamase E coli pBR322 origin Low copy replication and growth in E coli araC gene Encodes the regulatory protein for tight regulation of the Paap promoter Lee 1980 Schleif 1992 Continued on next page pBAD His Vector continued Map of pBAD His The figure below summarizes the features of the pBAD His vector Complete sequences for all three pBAD His vectors are available for downloading at www invitrogen com or by contacting Technical Support see page 28 Details of
6. cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD His A is available for downloading at www invitrogen com or from Technical Support see page 28 02 Region I AAGAAACCAA TTGTCCATAT TCTCGCTAAC CAAACCGGTA AAGCCATGAC AAAAACGCGT Saal ATTATTTGCA CGGCGTCACA 35 kar ATCCTACCTG ACGCTTTTTA EN RBS Neo TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT ACCCCGCT AACAAAAG TA TTAAAAGCAT TCTGTAACAA AGCGGGACCA O4 Region I G TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward CTTTGCTAT Ppriming site PG er TTTATCCATA AGATTAGCGG 12 and l4 Region 10 El TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTGGGC Polyhistidine Region I TAACAGGAGG AATTAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT Met Gly G AGC ATG ACT GGT GGA CAG CAA Al ly Ser His His His His His His Gly Met Ala mar EK recognition site Xpress Epitope l I TG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys EK cleavage site A Xhol Sac l Bg Il Pst Pvu ll Kpn EcoRI Sful GAT CGA TGG GGA TCC GAG LA A CTC GAG ATC TGC AGC TGG TAC CAT ATG GGA ATT CGA Asp Arg Trp Gly Ser Glu Leu Glu Ile Cys Ser Trp Tyr His Met Gly Ile
7. each multiple cloning site are shown on pages 9 11 TM ATG 6xHis En Comments for pBAD His A 4102 nucleotides Sac and Pvu Il are not present in version C araBAD promoter region bases 4 276 Initiation ATG bases 319 321 Polyhistidine tag bases 331 348 Xpress epitope bases 388 411 Enterokinase recognition site bases 397 411 Multiple cloning site bases 430 470 rrnB transcription termination region bases 553 710 Ampicillin ORF bases 989 1849 pBR322 origin bases 1994 2667 AraC ORF bases 4076 3198 pBAD Myc His Vector Features of The important elements of pBAD Myc His A 4094 bp pBAD Myc His B pBAD Myc His 4092 bp and pBAD Myc His C 4093 bp are described in the following table All features have been functionally tested Feature Benefit araBAD promoter Paap Provides tight dose dependent regulation of heterologous gene expression Guzman et al 1995 Optimized ribosome binding site Increases efficiency of recombinant fusion protein expression Initiation ATG Provides a translational initiation site for the fusion protein Multiple cloning site Allows insertion of your gene for expression C terminal myc epitope tag Allows detection of the fusion protein Glu GIn Lys Leu Ile Ser Glu Glu by the Anti Myc Antibody Asp Leu Evans et al 1985 see page vi for ordering information C terminal polyhistidine region Forms metal binding site for affinity purificat
8. with Nco I and Nsi I to remove the lacl4 gene and the trc promoter and replacing with an Nco I Nsi I fragment containing the araC gene and the araBAD promoter The P galactosidase portion of the fusion may be released by digestion with BamH l and Hind II The vector expresses a 120 kDa protein The figure below summarizes the features of the pBAD His lacZ vector The Control Vector complete nucleotide sequence for pBAD His lacZ is available at 23 www invitrogen com or by contacting Technical Support page 28 TM PBAD ATG 6xHis Ebitone Arne lac Z term pBAD His lacZ 7 1 kb Comments for pBAD His lacZ 7115 nucleotides araBAD promoter region bases 4 276 Initiation ATG bases 320 322 Polyhistidine tag bases 332 349 Xpress epitope bases 389 412 Enterokinase recognition site bases 398 412 LacZ ORF bases 419 3475 rrnB transcription termination region bases 3565 3722 Ampicillin ORF bases 4002 4862 pBR322 origin bases 5007 5680 AraC ORF bases 7089 6211 pBAD Myc His lacZ Description Map of Control Vector Comments for pBAD Myc His lacZ 7241 nucleotides pBAD Myc His lacZ is a 7242 bp control vector containing the gene for B galactosidase fused to the C terminal peptide It was constructed by digesting the vector pTrcHis2 lacZ with Nco I and NsiI to remove the lacl gene and the trc promoter and replacing with an Nco I Nsi I fragment containing the araC gene and the ara BAD promoter The B
9. 01 357 414 471 531 591 651 region The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD Myc His B is available for downloading at www invitrogen com or from Technical Support see page 28 02 Region AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA O4 Region AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward priming site ed ATTATTTGCA CGGCGTCACA CTTTGCTATG CCATACCATT TTTATCCATA AGATTAGCGG l2 and l4 Region 35 10 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTGGGC A RBS Nco Xhol Sac Bgl Il Pst Kpn lii l l TAACAGGAGG AATTAACC ATG GATCCGAGCT CGAGATCTGC AGCTGGTACC ATATG Met EcoRI Sful Hindill Xbal myc epitope l Io l GGAATTCGAA GCTTTCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT AGC GCC Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Polyhistidine Region GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTAAACGG TCTCCAGCTT GGCTGTTTTG Val Asp His His His His His His pBAD reverse priming site l GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT rrnB T4 and T9 transcriptional terminators CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG A
10. 7 C with shaking 225 250 rpm to ODsoo 1 2 The next day label five tubes 1 through 5 and add 10 ml of SOB or LB containing 50 pg ml ampicillin Inoculate each tube with 0 1 ml of the overnight culture Grow the cultures at 37 C with vigorous shaking to an ODgoo 0 5 the cells should be in mid log phase While the cells are growing prepare four 10 fold serial dilutions of 20 L arabinose with sterile water and aseptic e g 2 0 2 0 02 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 Add L arabinose to the five 10 ml cultures as follows Tube Volume ml Stock Solution Final Concentration 1 0 1 0 002 0 00002 2 0 1 0 02 0 0002 3 0 1 0 2 0 002 4 0 1 2 0 02 5 0 1 20 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 Continued on next page Expression continued Preparation of Samples Analysis of Samples Low Expression pBAD sequencing primers Before starting prepare SDS PAGE gels to analyze all the samples you collected Note If you wish to analyze your samples for soluble protein see the next page for a protocol 1 When all the samples have been collected from Steps 8 and 11 previous page resuspend each pelle
11. ATTAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT Met Gly Gly Ser His His His His His His Gly Met Ala EK recognition site Xpress Epitope I 358 AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys EK cleavage site Xho Sacl Bg Il Pst Pvu ll Kpn EcoR Sful Hind Ill eN nern I cd l 412 GAT CCG AGC TCG AGA TCT GCA GCT GGT ACC ATA TGG GAA TTC GAA GCT TGG Asp Pro Ser Ser Arg Ser Ala Ala Gly Thr Ile Trp Glu Phe Glu Ala Trp pBAD reverse priming site 463 CTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA 521 AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC rrnB T4 and Ta transcriptional terminator 581 ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG 641 AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT 701 TCGTTTTATC TGTTGTTTG Continued on next page 10 Cloning into pBAD His continued pBAD His C Below is the multiple cloning site for pBAD His C Restriction sites are labeled to Multiple Cloning indicate the cleavage site The boxed nucleotides indicate the variable region The Site multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD His C is available for downloading at www invitrogen com or from Technical Support see page 28
12. Arg Hind Ill l AGC TTG GCTGTTTTG GCGGAT Ser Leu AGCGGTCTGA TAAAACAGAA pBAD reverse priming site GAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA rrnB T4 and To transcriptional terminator TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC TCGTTTTAT ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT Continued on next page Cloning into pBAD His continued pBAD His B Below is the multiple cloning site for pBAD His B Restriction sites are labeled to Multiple Cloning indicate the cleavage site The boxed nucleotides indicate the variable region The Site multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD His B is available for downloading at www invitrogen com or from Technical Support see page 28 02 Region 1 AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT 61 TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA O4 Region I 121 AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward priming site al 181 ATTATTTGCA CGGCGTCACA CTTTGCTATG aan TTTATCCATA AGATTAGCGG 12 and l4 Region 35 10 241 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTGGGC el RBS Neo Polyhistidine Region End l l 301 TAACAGGAGG A
13. GAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC Continued on next page 14 Cloning into pBAD Myc His continued pBAD Myc His C Below is the multiple cloning site for pBAD Myc His C Restriction sites are Multiple Cloning labeled to indicate the cleavage site The boxed nucleotides indicate the variable Site 61 121 181 241 301 362 415 472 532 592 652 15 region The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD Myc His C is available for downloading at www invitrogen com or from Technical Support see page 28 O7 Region l AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA O4 Region I AAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTG CAP binding site pBAD forward priming site zn ATTATTTGCA CGGCGTCACA CTTTGCTATG Brenn TTTATCCATA AGATTAGCGG l4 and l2 Region 35 10 ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TITTITGGGC gt RBS Nco Xhol Sac1Bgl l Pst Kpn EcoR ER ren Feen TAACAGGAGG AATTAACC ATG GATCCGAGCT CGAGATCTGC AGCTGGTACC ATATGGGAAT Met Sfu Hind Ill a l myc epitope TCGAAGCTITA CGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT cra AAT AGC GCC Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Polyhistidine Reg
14. ame at the 5 end Be sure to include a stop codon to terminate translation of your protein The multiple cloning sites of each version of pBAD His are provided on pages 9 11 Restriction sites are labeled to indicate cleavage site The boxed sequence is the variable region that facilitates in frame cloning with the N terminal peptide This variable region is located between the enterokinase cleavage site and the Xho 1 site Features of the araBAD and araC promoters are marked and described as follows For more information see Lee 1980 Miyada et al 1984 Lee et al 1987 and Schleif 1992 e _O region Binding site of AraC that represses transcription from Pap e O1 region Binding site of AraC that represses transcription of the araC promoter Pc transcribed on the opposite strand not shown e CAP binding site Site where CAP cAMP binding protein binds to help activate transcription from Pap and Pc e land Ii regions Binding sites of AraC that activate transcription from Paap e 10and 35 regions Binding sites of RNA polymerase for transcription of PBAD Each multiple cloning site has been confirmed by sequencing and functional testing Continued on next page Cloning into pBAD His continued pBAD His A Multiple Cloning Site 61 121 181 241 301 358 412 466 531 591 651 711 Below is the multiple cloning site for pBAD His A Restriction sites are labeled to indicate the
15. ated between the multiple cloning site and the myc epitope Features of the araBAD and araC promoters are marked and described as follows For more information see Lee 1980 Miyada et al 1984 Lee et al 1987 and Schleif 1992 e Op region Binding site of AraC that represses transcription from Pap e Oi region Binding site of AraC that represses transcription of the araC promoter Pc transcribed on the opposite strand not shown e CAP binding site Site where CAP cAMP binding protein binds to help activate transcription from Paap and Pc e land l regions Binding sites of AraC that activate transcription from Paap e 10and 35 regions Binding sites of RNA polymerase for transcription of PBAD Each multiple cloning site has been confirmed by sequencing and functional testing Continued on next page 12 Cloning into pBAD Myc His continued pBAD Myc His A Below is the multiple cloning site for pBAD Myc His A Restriction sites are Multiple Cloning labeled to indicate the cleavage site The boxed nucleotides indicate the variable Site region The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pBAD Myc His A is available for downloading at www invitrogen com or from Technical Support see page 28 02 Region I 1 AAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT 61 TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCA
16. dium is stable for only 1 2 weeks 1X M9 Salts See next page for recipe for 10X M9 Salts 2 Casamino Acids 0 2 glucose 1 mM MsCh 50 100 ug 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 After the autoclaved solution has cooled add the following sterile solutions aseptically 4 10X M9 Salts 100 ml 5 1MMgCh 1 ml 6 20 glucose 10 ml 7 100 mg ml ampicillin 0 5 to 1 ml 8 Mix well and store medium containing ampicillin at 4 C Medium is good for 1 month at 4 C Continued on next page 26 Recipes continued 10X M9 Salts Lysis Buffer 27 For 1 liter Na gt HPO4 60 8 KH2PO4 30 g NaCl 58 NH4CI 10 g Water 900 ml 1 Dissolve reagents in the water and adjust the pH to 7 4 with 10 M NaOH 2 Add water to 1 liter and autoclave for 20 minutes on liquid cycle 3 Cool and add 1 ml of 1 M thiamine filter sterilize Store at room temperature 10 mM Tris HCl pH 8 1mMEDTA 0 5 mg ml lysozyme 0 1 mg ml DNase I 10 mM CaClo 1 Prepare just before use Take 10 ml of TE buffer and add 5 mg of lysozyme 1 mg of DNase I and 0 1 ml of 1 M CaCL 2 Gently mix and store on ice Use immediately 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 formu
17. e the pellets on ice see Step 5 4 Mix together equal amounts of supernatant and 2X SDS Sample buffer and heat for 5 minutes at 70 C 5 Add 200 ul of 1X SDS PAGE sample buffer to pellets from Step 3 and heat for 5 minutes at 70 C 6 Load 10 ul of the supernatant sample and 10 ul of the pellet sample onto an SDS PAGE and electrophorese 7 Analyze for optimal soluble expression of your protein To ensure low levels of expression you may find it useful to utilize glucose to repress the araBAD promoter further Follow the steps below to express your protein e Transform your construct into LMG194 LMG194 can be grown in RM medium which enables repression of P sap by glucose e Follow the Pilot Expression on page 19 substituting RM Medium Glucose medium see page 26 to grow the cells e Be sure to monitor the ODso as the cells will grow more slowly in RM medium e Induce with various concentrations of L arabinose as described in the Pilot Expression e Monitor OD go over time be sure cells are growing Purification Scale up of Expression for Purification Purification We recommend using the ProBond Purification System available separately from Invitrogen see page vi for ordering information Use the conditions determined in the previous section to grow and induce 50 ml of cells This is the largest culture volume to use with the 2 ml prepacked columns included in the ProBond Purification S
18. expression it is helpful to vary the L arabinose concentration and or run a time course of expression to determine the best conditions for optimal expression of your particular protein A mock expression consisting of the pBAD His or pBAD Myc His vector alone should be done as a negative control pBAD His lacZ or pBAD Myc His lacZ are included for use as positive expression controls see pages 23 24 TOP10 may be used as a general host for expression LMG194 should be used if your protein is toxic or essential to E coli Basic Strategy Once you have some clones that you wish to characterize we recommend the following strategy to determine the optimal expression level 1 Pilot Expression In this expression experiment you will vary the amount of L arabinose over a 10 000 fold range 0 00002 to 0 2 to determine the approximate amount of L arabinose needed for maximum expression of your protein See next page for protocol 2 To optimize expression of your protein you may wish to try L arabinose concentrations spanning the amount determined in Step 1 Or you may wish to perform a time course Note If your expressed protein is insoluble remember to analyze the supernatant and the pellet of lysed cells for expression of soluble protein Note If you transformed your pBAD His or pBAD Myc His construct into LMG194 be sure to perform your expression experiments in RM medium with glucose see page 26 for recipe to ensure low basal levels of yo
19. galactosidase portion of the fusion may be released by digestion with Sfu I BstB I Other cloning options are possible The vector expresses a 120 kDa protein The figure below summarizes the features of the pBAD Myc His lacZ vector The complete nucleotide sequence for pBAD Myc His lacZ is available at www invitrogen com or by contacting Technical Support see page 28 pBAD Myc His lacZ 7 2 kb araBAD promoter region bases 4 276 Initiation ATG bases 319 321 LacZ ORF bases 373 3429 myc epitope bases 3523 3552 Polyhistidine tag bases 3568 3585 rrnB transcription termination region bases 3691 3848 Ampicillin ORF bases 4128 4988 pBR322 origin bases 5133 5806 AraC ORF bases 7215 6337 24 Recipes Pre mixed Media Low Salt LB Medium with Ampicillin Low Salt LB Agar Plates with Ampicillin 25 Invitrogen carries pre mixed growth media such as imMedia in convenient pouches or in bulk imMedia is pre mixed and pre sterilized for convenient preparation of liquid medium or agar plates for E coli growth and is available with or without IPTG and X gal and a choice of three antibiotics ampicillin kanamycin or Zeocin selection agent Refer to page vii for ordering information LB Medium per liter 1 Tryptone 0 5 Yeast Extract 0 5 NaCl pH7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 5 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution
20. ications K950 01 Purification Columns 50 R640 50 10 ml polypropylene columns EKMax Enterokinase 250 units E180 01 Continued on next page Additional Products continued Competent E coli Pre mixed Media For your convenience TOP10 is available as electrocompetent or chemically competent cells in a One Shot kit format For more information refer to www invitrogen com or contact Technical Support page 28 Item Quantity Cat No Electrocomp TOP10 20 reactions C664 55 2 x 20 reactions C664 11 6 x 20 reactions C664 24 One Shot TOP10 Competent Cells 20 reactions C4040 03 Invitrogen carries pre mixed growth media in convenient pouches or in bulk See the table below for ordering information Item Quantity Cat No imMedia Amp Liquid 20 pouches Q600 20 200 ml medium imMedia Amp Agar 20 pouches 8 10 plates Q601 20 Ampicillin Sodium Salt irradiated 200 mg 11593 027 vil Overview Introduction Regulation of Expression by L arabinose Introduction The pBAD His and pBAD Myc His plasmids are pBR322 derived expression vectors designed for regulated dose dependent recombinant protein expression and purification in E coli Optimum levels of soluble recombinant protein are possible using the araBAD promoter Paap from E coli The regulatory protein AraC is provided on the pBAD His and pBAD Myc His vectors allo
21. invitrogen pBAD His A B and C pBAD Myc His A B and C Vectors for Dose Dependent Expression of Re combinant Proteins Containing N or C Terminal 6xHis Tags in E coli Catalog nos V430 01 V440 01 Version J 29 December 2010 25 0187 ii Table of Contents Kit Contents and Storage uunenenenssenennnennnnnsenennnnnnsnensnnnnnnnnnnnnsnnnnnnenennnnnnnnnnnnnsnnennnnnsnsensnnesnsnsnnennenenenn v Additional Prod S A e 222222 AS Ian vi IntrOodUCHON En 1 OVERVIEW O A A A SA r entente baa er 1 PBAD His Veet r 2 222222 AAA A E edn 3 PBAD Myc His Vector Aa hye n e a Reiner nina 5 Method CE 7 General ClONiINg it a a 7 Cloning into pBAD HlS acuario piola alada 8 Cloning into pBAD Myo A sHih knnnbieslhnniie 12 Eis COMM APANSLOLIVATOM it A RR ie A AAA EA AAA AAA aie 16 EXPLeSSlON Rae ee 17 PUE CAMION A rn an eet len eee entel SR eters lee 22 PDDONGIX a dad dell dadde dua dadde edn 23 PBAD His Mall oi a ara ER un une Riesa 23 pBAD Myc Hlis lacio venusta rennen 24 O NN 25 Technical SUPPO sss eis tess acess HERR elende den Lasse les seas Dea toda dis RE 28 Purchaser NOt fica th nie ia 29 RELEREN CES oases A a toe state Be elle 30 iii iv Kit Contents and Storage Contents This manual is supplied with the following kits Cat No Contents V430 01 20 ug each of pBAD His A B and C vector in TE Buffer pH 8 0 40 ul at 0 5 ug ul 20 ug each of pBAD His lacZ vector in TE Buffer pH 8 0 40 ul at 0 5 ug
22. ion GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTAAACGG TCTCCAGCTT GGCTGTTTTG Val Asp His His His His His His pBAD reverse priming site GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGA AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT rrnB T4 and Tg transcriptional terminators CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC E coli transformation E coli Transformation Glycerol Stock After ligating your insert into the appropriate vector transform your ligation mixtures into TOP10 and select on LB plates containing 50 100 pg ml ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert Once you have obtained your desired construct we recommend that you store your clone as a glycerol stock 1 Grow 1 to 2 ml of the strain containing your construct in pBAD His or pBAD Myc His to saturation 12 16 hours ODsoo 1 2 in LB containing 50 100 pg ml ampicillin Combine 0 85 ml of the culture with 0 15 ml of sterile glycerol Mix the solution by vortexing Transfer to an appropriate vial for freezing and cap aD Freeze in an ethanol dry ice bath or liquid nitrogen and then transfer to 80 C for long term storage 16 Expression Introduction Since each recombinant protein has different characteristics that may affect optimum
23. ion of recombinant fusion protein on metal chelating resin In addition it allows detection of the recombinant protein with Anti His C term antibodies and the Penta His Mouse IgG1 Monoclonal Antibody see page vi for ordering information rrnB transcription termination region Strong transcription termination region Ampicillin resistance gene Allows selection of the plasmid in B lactamase E coli pBR322 origin Low copy replication and growth in E coli araC gene Encodes the regulatory protein for tight regulation of the Paap promoter Lee 1980 Schleif 1992 Continued on next page pBAD Myc His Vector continued Map of pBAD The figure below summarizes the features of the pBAD Myc His vector Myc His Complete sequences for all three pBAD Myc His vectors are available for downloading at www invitrogen com or by contacting Technical Support see page 28 Details of each multiple cloning site are shown on pages 13 15 ATG MCS myc 6xHis pBAD Myc His A B C 4 1 kb Comments for pBAD Myc His A 4094 nucleotides araBAD promoter region bases 4 276 ser he A a Initiation ATG bases 319 321 Multiple cloning site bases 317 370 Version B contains Xba only myc epitope bases 377 406 Polyhistidine tag bases 422 439 rrnB transcription termination region bases 545 702 Ampicillin ORF bases 981 1841 pBR322 origin bases 1986 2659 AraC ORF bases 4068 3190 Version C c
24. lations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com MSDS Certificate of Analysis Limited Warranty E mail eurotech invitrogen com Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Invitrogen 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 prod
25. maintain the empty vectors and recombinant 7 constructs transform them into a recA endA E coli host i e TOP10 3 Ligate your gene of interest into pBAD His or pBAD Myc His 16 transform into TOP10 or LMG194 and select on 50 100 pg ml ampicillin 4 Sequence your construct to ensure that it is in frame with the 16 N terminal pBAD His or C terminal pBAD Myc His peptide 5 Perform a 4 hour expression using a 10 000 fold range of 17 L arabinose concentrations e g 0 00002 0 0002 0 002 0 02 and 0 2 Use appropriate controls Vectors expressing B galactosidase are available with each kit Antibodies are available for detection of recombinant proteins see page vi for ordering information 6 Optimize expression by varying L arabinose concentration or 21 the time of induction 7 Purify your recombinant protein by chromatography on a metal 22 chelating resin see page vi for ordering information pBAD His Vector Features of The important elements of pBAD His A 4102bp pBAD His B 4092 bp and pBAD His pBAD His C 4100 bp are described in the following table All features have been functionally tested Feature Benefit araBAD promoter Pap Provides tight dose dependent regulation of heterologous gene expression Guzman et al 1995 Optimized ribosome binding site Increases efficiency of recombinant fusion protein expression Initiation ATG
26. ness for a particular purpose 28 Purchaser Notification Limited Use Label This product is licensed under U S and foreign patents from Hoffmann License No 22 LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland Vectors Clones and is provided only for use in research Information about licenses for commer Encoding cial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Histidine Hexamer Germany 29 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 Bachmann B J 1990 Linkage map of Escherichia coli K 12 edition 8 Microbiol Rev 54 130 197 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 15931 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 Guzman L M Barondess J J and Beckwith J 1992 FtsL
27. ontains SnaB only Methods General Cloning Introduction E coli Host Genotype of TOP10 Genotype of LMG194 Maintenance of pBAD His and pBAD Myc His The following information is provided to help you clone your gene of interest into pBAD His or pBAD Myc His For basic information on DNA ligations E coli transformations restriction enzyme analysis DNA sequencing and DNA biochemistry see Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 For cloning and transformation we recommend using a recA endA strain such as TOP10 included in the kit also available separately see page vii This strain is capable of transporting L arabinose but not metabolizing it This is important for expression studies as the level of L arabinose will be constant inside the cell and not decrease over time Please note that other strains may be suitable for general use Be sure to check the genotype of your strain It should be araBADC and araEFGH Bachmann 1990 The E coli strain LMG194 is included in the kit to ensure low basal level expression of toxic genes Guzman et al 1995 This strain is capable of growth on minimal medium RM medium which allows additional repression of Psap by glucose Once you have determined that you have the correct construct transform it into LMG194 prior to performing expression experiments F merA A mrr hsdRMS mc
28. rBC 80lacZAM15 AlacX74 recAl araD139 A araA leu 7697 galU galK rpsL endA1 nupG Note This strain is araBADC It is deleted for both araBA and araC and the gene for araD has a point mutation in it making it inactive F AlacX74 gal E thi rpsL AphoA Pvu II Aara714 leu Tn10 Please note that this strain is streptomycin and tetracycline resistant To propagate and maintain pBAD His or pBAD Myc His use the supplied 0 5 ug pl stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10F DH5a T1 R TOP10 or equivalent Select transformants on LB plates containing 50 100 pg ml ampicillin Note Use strains like DH5a only for propagation of pBAD His or pBAD Myc His but not expression of recombinant proteins see explanation above Be sure to prepare a glycerol stock of each plasmid for long term storage see page 16 Cloning into pBAD His Important pBAD His Multiple Cloning Sites To generate recombinant proteins that are expressed correctly and contain the N terminal fusion peptide it is necessary to clone in frame with the N terminal peptide To facilitate cloning the pBAD His vector is provided in three different reading frames They differ only in the spacing between the sequences that code for the N terminal peptide and the multiple cloning site For proper expression first determine which restriction sites are appropriate for ligation and then which vector will preserve the reading fr
29. rbor 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 2008 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 30 invitrogen Corporate Headquarters Invitrogen Corporation 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
30. rmation refer to www invitrogen com or contact Technical Support page 28 Expression of your recombinant protein can be detected using an antibody to the appropriate epitope The table below describes the antibodies available for use with pBAD His or pBAD Myc His Horseradish peroxidase HRP conjugated antibodies allow one step detection using colorimetric or chemiluminescent detection methods Vector Epitope Antibody Cat No pBAD His Anti Xpress Anti Xpress R910 25 Anti Xpress HRP Antibody R911 25 N terminal Penta His Mouse IgG1 P21315 polyhistidine Monoclonal Antibody tag pBAD Myc His c myc Anti Myc R950 25 Anti Myc HRP R951 25 C terminal Anti His C term R930 25 Fi Anti His C term HRP R931 25 Anti His C term AP Antibody R932 25 Penta His Mouse IgG1 P21315 Monoclonal Antibody The metal binding domain encoded by the polyhistidine tag allows simple easy purification of your recombinant protein by Immobilized Metal Affinity Chromatography IMAC while EKMax Enterokinase allows removal of the N terminal peptide for production of native protein See the table below for ordering information Product Quantity Cat No ProBond Purification System 6 purifications K850 01 ProBond Metal Binding Resin precharged resin 50 ml R801 01 provided as a 50 slurry in 20 ethanol 150 m1 R801 15 Ni NTA Purification System 6 purif
31. rward primer 5 ATGCCATAGCATTTITATCC 3 pBAD reverse primer 5 GATTTAATCTGTATCAGG 3 Continued on next page 20 Expression continued Optimization of Expression Preparation of Samples for Soluble Insoluble Protein Expression of Toxic Proteins 21 Once you have detected expression of your protein you may wish to perform some experiments to further optimize expression Use the Pilot Expression protocol but vary the L arabinose concentration over a smaller range For example if you obtained the best expression at 0 002 try 0 0004 0 0008 0 001 0 004 and 0 008 Also you may perform a time course of induction over a 5 to 6 hour time period taking time points every hour to determine if varying the time increases expression If your protein is insoluble you may wish to analyze the supernatant and pellet of lysed cells when you vary the L arabinose concentration Refer to the protocol on the next page to prepare samples Remember to store your time points at 20 C After collecting all of your samples prepare SDS PAGE gels for analysis 1 When all the samples have been collected thaw and resuspend each pellet in 100 ul of Lysis Buffer see Recipes page 27 2 Place sample on ice and sonicate the solution for 10 seconds 3 Centrifuge samples in a microcentrifuge at maximum speed for 1 minute at 4 C to pellet insoluble proteins Transfer supernatant to a fresh tube and store on ice Stor
32. t in 100 ul of 1X SDS PAGE sample buffer Boil 5 minutes and centrifuge briefly Load 5 ul of each sample on an SDS PAGE gel and electrophorese Save your samples by storing at 20 C 1 Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein 2 Use a negative control empty vector to distinguish recombinant proteins from background proteins 3 Use the positive control pBAD His lacZ or pBAD Myc His lacZ to confirm that growth and induction was done properly The positive control should yield a 120 kDa protein 4 You should be able to determine the approximate L arabinose concentration for maximum expression If you don t see any expression on a Coomassie stained gel re run your samples on an SDS PAGE gel and perform a western blot Use antibody to your protein or one of the recommended antibodies appropriate for your protein See page vi for ordering information For more information refer to www invitrogen com or contact Technical Support page 28 If you still don t see expression of your protein sequence your construct and make sure it is in frame with the N or C terminal peptide You may use the pBAD forward and reverse sequencing primers to sequence your insert containing your gene of interest in pBAD His or pBAD Myc His vectors to make sure that it is in frame with the N or C terminal peptide Primer Sequence pBAD fo
33. to 7 0 with 5 M NaOH and bring the volume to 1 liter 3 Autoclave for 20 minutes on liquid cycle Let solution cool to 55 C Add ampicillin to a final concentration of 50 ug ml Store the medium at 4 C Medium is stable for only 1 2 weeks LB Medium per liter 1 Tryptone 0 5 Yeast Extract 0 5 NaCl 1 5 Agar pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 5 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution to 7 0 with 5 M NaOH add 15 g agar and bring the volume to 1 liter 3 Autoclave for 20 minutes on liquid cycle Let agar cool to 55 C Add ampicillin to a final concentration of 50 pg ml Pour into 10 cm petri plates Let the plates harden then invert and store at 4 C Plates containing ampicillin are stable for 1 2 weeks Continued on next page Recipes continued SOB Medium with Ampicillin RM Medium Glucose SOB per liter 2 Tryptone 0 5 Yeast Extract 0 05 NaCl 2 5 mM KC 10 mM MgCl 1 Dissolve 20 g tryptone 5 g yeast extract and 0 5 g NaCl in 950 ml deionized water 2 Make a 250 mM KCI solution by dissolving 1 86 g of KCl in 100 ml of deionized water Add 10 ml of this stock KCI solution to the solution in Step 1 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 ug ml 5 Store at 4 C Me
34. ucts and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will 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 Invitrogen Corporation s liability only to the cost 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 Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen 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 Invitrogen 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 Invitrogen assumes 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 fit
35. ul 1 ml sterile 20 L arabinose 1 stab LMG194 1 stab TOP10 V440 01 20 ug each of pBAD Myc His A B and C vector in TE Buffer pH 8 0 40 ul at 0 5 ug ul 20 ug each of pBAD Myc His lacZ vector in TE Buffer pH 8 0 40 ul at 0 5 ug pl 1 ml sterile 20 L arabinose 1 stab LMG194 1 stab TOP10 TE Buffer pH 8 0 10 mM Tris HCI 1 mM EDTA pH 8 0 Shipping Storage Kits are shipped on wet ice Upon receipt store the plasmids and the 20 L arabinose solution at 20 C and the stabs at 4 C Long Term For long term storage of E coli strains supplied as stabs with this kit prepare Storage glycerol stocks as follows 1 Grow the E coli strain overnight in LB medium overnight with antibiotic selection when appropriate 2 Combine 0 85 ml of the overnight culture with 0 15 ml of sterile glycerol 3 Vortex and transfer to a labeled cryovial 4 Freeze the tube in liquid nitrogen or dry ice ethanol bath and store at 80 C Note Grow LMG194 strain in RM medium containing M9 salts see page 26 27 Additional Products Accessory Products Detection of Recombinant Proteins Purification of Recombinant Proteins vi Invitrogen offers a variety of products that are suitable for use with the pBAD His and pBAD Myc His plasmids Ordering information is provided below For detailed instructions on how to use any of the accessory products refer to the manual provided with each product For more info
36. ur protein Expression of your protein with N or C terminal tags will increase the size of your protein Refer to the table below for the approximate size of the N and C terminal tags Be sure and account for any additional amino acids between the tag and your protein Note Vector Tag Size pBAD His N terminal Anti Xpress tag 3 kDa pBAD Myc His C terminal Myc His tag 2 kDa Continued on next page 17 Expression continued Before Starting Be sure to have the following solutions and equipment on hand before starting the experiment e SOB or LB containing 50 ug ml ampicillin see Recipes pages 25 26 RM medium containing glucose see Recipes page 26 e 37 C shaking incubator e 20 L arabinose provided e 37 C heat block or water bath e 42 C water bath e Liquid nitrogen e 1X and 2X SDS PAGE sample buffer e Reagents and apparatus for SDS PAGE gel e 70 C water bath e Lysis Buffer see page 27 for recipe e Sterile water Continued on next page 18 Expression continued Pilot Expression 19 Remember to include the appropriate negative and positive controls to evaluate your expression experiment 1 For each transformant or control inoculate 2 ml of SOB or LB medium containing 50 pg ml ampicillin with a single recombinant E coli colony If you are using LMG194 as a host use RM medium containing glucose and 50 100 pg ml ampicillin Grow overnight at 3
37. wing regulation of Prap In the presence of L arabinose expression from Pap is turned on while the absence of L arabinose produces very low levels of transcription from Paap Lee 1980 Lee et al 1987 Uninduced levels are repressed even further by growth in the presence of glucose Glucose reduces the levels of 3 5 cyclic AMP thus lowering expression of the catabolite repressed Paap promoter Miyada et al 1984 By varying the concentration of L arabinose protein expression levels can be optimized to ensure maximum expression of soluble 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 refer to Schleif 1992 Continued on next page Overview continued Experimental The table below describes the basic steps needed to clone and express your Outline protein using pBAD His or pBAD Myc His For more details refer to the page s indicated Step Action Page 1 Develop a cloning strategy to ligate your gene of interest into the 7 desired vector Refer to the appropriate pages for the multiple cloning sites of each version of the vector pBAD His A B and C 8 11 pBAD Myc His A B and C 12 15 2 To propagate and
38. ystem If you need to purify larger amounts of recombinant protein you may need more ProBond resin Note Remember to use RM medium page 26 with LMG194 1 Inoculate 2 ml of SOB or LB medium containing 50 pg ml ampicillin with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 250 rpm to ODsoo 1 2 The next day inoculate 50 ml of SOB or LB medium containing 50 pg ml ampicillin with 1 ml of the overnight culture 4 Grow the culture at 37 C with vigorous shaking to an ODgoo 0 5 the cells should be in mid log phase Add the optimal amount of L arabinose to induce expression Grow at 37 C with shaking until the optimal time point is reached Harvest the cells by centrifugation 3000 x g for 10 minutes at 4 C 7 At this point you may proceed directly to purification or store at 80 C for future use TM For help with purification of your recombinant protein refer to the ProBond Purification System manual See page vi for other recommended purification products available from Invitrogen For more information refer to www invitrogen com or contact Technical Support page 28 If you are using another type of resin follow manufacturer s recommendations 22 Appendix pBAD His lacZ Description Map of pBAD His lacZ is a 7115 bp control vector containing the P galactosidase gene fused to the N terminal peptide It was constructed by digesting the vector pTrcHis lacZ
Download Pdf Manuals
Related Search