pBAD/Myc - Gene Synthesis

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Cloning into pBAD/HisImportantTo generate recombinant proteins that are expressed correctly and contain the N-terminalfusion peptide, it is necessary to clone in frame with the N-terminal peptide. To facilitatecloning, the pBAD/His vector is provided in three different reading frames. They differonly in the spacing between the sequences that code for the N-terminal peptide and themultiple cloning site. For proper expression, first determine which restriction sites areappropriate for ligation and then which vector will preserve the reading frame at the 5´end. Be sure to include a stop codon to terminate translation of your protein.pBAD/HisMultiple CloningSitesThe 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 variableregion that facilitates in frame cloning with the N-terminal peptide. This variable region islocated between the enterokinase cleavage site and the Xho I site.Features of the araBAD and araC promoters are marked and described as follows. Formore information please see Lee, 1980; Miyada, et al., 1984; Lee, et al., 1987; andSchleif, 1992.• O 2 region: Binding site of AraC that represses transcription from P BAD.• O 1 region: Binding site of AraC that represses transcription of the araC promoter (P C)(transcribed on the opposite strand; not shown).• CAP binding site: Site where CAP (cAMP binding protein) binds to help activatetranscription from P BAD and P C.• I 2 and I 1 regions: Binding sites of AraC that activate transcription from P BAD.• -10 and -35 regions: Binding sites of RNA polymerase for transcription of P BAD.Each multiple cloning site has been confirmed by sequencing and functional testing.continued on next page8
Cloning into pBAD/His, continuedpBAD/His AMultiple Cloning Site1O 2 RegionAAGAAACCAA TTGTCCATAT TGCATCAGAC ATTGCCGTCA CTGCGTCTTT TACTGGCTCT61121181241301358412466TCTCGCTAAC CAAACCGGTA ACCCCGCTTA TTAAAAGCAT TCTGTAACAA AGCGGGACCAO 1 RegionAAGCCATGAC AAAAACGCGT AACAAAAGTG TCTATAATCA CGGCAGAAAA GTCCACATTGCAP binding siteATTATTTGCA CGGCGTCACA CTTTGCTATG CCATAGCATT TTTATCCATA AGATTAGCGGI 2 and I 1 Region-35 -10ATCCTACCTG ACGCTTTTTA TCGCAACTCT CTACTGTTTC TCCATACCCG TTTTTTGGGCRBSNco IPolyhistidine RegionTAACAGGAGG AATTAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCTMet Gly Gly Ser His His His His His His Gly Met AlaXpress EK recognition siteEpitopeAGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAGSer Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp LysEK cleavage siteXho I Sac I Bgl II Pst I Pvu II Kpn I EcoR I Sfu IGAT CGA TGG GGA TCC GAG CTC GAG ATC TGC AGC TGG TAC CAT ATG GGA ATT CGAAsp Arg Trp Gly Ser Glu Leu Glu Ile Cys Ser Trp Tyr His Met Gly Ile ArgHind IIIAGC TTG GCTGTTTTG GCGGATGAGA GAAGATTTTC AGCCTGATAC AGATTAAATC AGAACGCAGASer Leu531591AGCGGTCTGA TAAAACAGAA TTTGCCTGGC GGCAGTAGCG CGGTGGTCCC ACCTGACCCCrrnB T1 and T2 transcriptional terminatorATGCCGAACT CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG651AGAGTAGGGA ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT711TCGTTTTATcontinued on next page9
Page 3 and 4: Table of ContentsTable of Contents.
Page 5 and 6: IntroductionOverviewIntroductionThe
Page 7 and 8: pBAD/His VectorFeatures ofpBAD/HisT
Page 9 and 10: pBAD/Myc-His VectorFeatures ofpBAD/
Page 11: MethodsGeneral CloningIntroductionT
Page 15 and 16: Cloning into pBAD/His, continuedpBA
Page 17 and 18: Cloning into pBAD/Myc-His, continue
Page 19 and 20: Cloning into pBAD/Myc-His, continue
Page 21 and 22: ExpressionIntroductionSince each re
Page 23 and 24: Expression, continuedPreparation of
Page 25 and 26: PurificationScale-up ofExpression f
Page 27 and 28: pBAD/Myc-His/lacZDescriptionpBAD/My
Page 29 and 30: Recipes, continuedSOB Medium(with A
Page 32: \ha\jZsZ_lky b kh[bjZ_l aZdZau gZ l

pBAD/Myc - Gene Synthesis

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