The pMAL-4 vectors (Figure 1) provide a method for expressing and purifying a protein produced from a cloned gene or open reading frame. The cloned gene is inserted downstream from the malE gene of E. coli, which encodes maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein (1,2). The MBP in these vectors has been engineered for tighter binding to amylose. The method uses the strong “tac” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences (3,4), and a one-step purification of the fusion protein using MBP’s affinity for maltose (5). The vectors express the malE gene (with or without its signal sequence) fused to the lacZα gene. Restriction sites between malE and lacZα are available for inserting the coding sequence of interest. Insertion inactivates the β-galactosidase α-fragment activity of the malE-lacZα fusion, which results in a blue to white color change on Xgal plates when the construction is transformed into an α-complementing host such as TB1, JM107 or NEB 5-alpha Competent E. coli (6,7). When present, the signal peptide on pre-MBP directs fusion proteins to the periplasm. For fusion proteins that can be successfully exported, this allows folding and disulfide bond formation to take place in the periplasm of E. coli, as well as allowing purification of the protein from the periplasm (8). The vectors carry the lacIq gene, which codes for the Lac repressor. This keeps expression from Ptac low in the absence of IPTG induction. The pMAL-4 vectors also contain the sequence coding for the recognition site of a specific protease, located just 5´ to the polylinker insertion sites. This allows MBP to be cleaved from the protein of interest after purification. The pMAL-c4X and pMAL-p4X vectors that are included in the system encode the site for Factor Xa (9, 10). Factor Xa cleaves after its four amino acid recognition sequence, so that few or no vector-derived residues are attached to the protein of interest, depending on the site used for cloning. pMAL vectors containing sites for alternative proteases are also available (Figure 1). The vectors pMAL-c4G (NEB #N8106) and pMAL-p4G (NEB #N8103) encode the site for GenenaseI (NEB #P8075), which cleaves following the sequence His-Tyr. The vectors pMAL-c4E (NEB #N8105) and pMAL-p4E (NEB #N8102) encode the site for Enterokinase (NEB #P8070), which cleaves following the sequence Asp-Asp-Asp-Asp-Lys.
| 载体基本信息 | |||
| 载体名称 | pMAL-p4x | 载体抗性 | Ampicillin |
|---|---|---|---|
| 载体长度 | 6720 bp | 载体类型 | E. coli Expression Vectors |
| 拷贝数 | High copy number | 启动子 | Tac |
| 克隆方法 | Enzyme digestion and ligation | 5引物 | MalE: 5-GGTCGTCAGACTGTCGATGAAGCC-3; MBP-F: 5-gatgaagccctgaaagacgcgcag-3 |
| 3引物 | pBad-R: 5-gatttaatctgtatcagg-3; M13-F: 5-TGTAAAACGACGGCCAGT-3 | 载体标签 | N-MBP, N-Factor Xa |
pMAL-p4x 载体载体图谱
pMAL-p4x 载体载体序列
LOCUS Exported 6720 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6720)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..6720
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 3..80
/gene="lacI (mutant)"
/note="lacIq promoter"
/note="In the lacIq allele, a single base change in the
promoter boosts expression of the lacI gene about 10-fold."
CDS 81..1163
/codon_start=1
/gene="lacI"
/product="lac repressor"
/note="lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
misc_feature 1406..1434
/note="tac promoter"
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 1442..1458
/bound_moiety="lac repressor encoded by lacI"
/note="lac operator"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
CDS 1528..2703
/codon_start=1
/gene="malE"
/product="maltose binding protein from E. coli"
/note="MBP"
/note="This gene encodes a signal sequence that can direct
MBP to the bacterial periplasm."
/translation="MKIKTGARILALSALTTMMFSASALAKIEEGKLVIWINGDKGYNG
LAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAE
ITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDK
ELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV
DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPS
KPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKD
PRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT"
CDS 2752..2763
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/note="Factor Xa site"
/translation="IEGR"
primer_bind complement(2811..2827)
/note="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
terminator 3176..3262
/gene="Escherichia coli rrnB"
/note="rrnB T1 terminator"
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 3354..3381
/note="rrnB T2 terminator"
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 3400..3491
/gene="bla"
/note="AmpR promoter"
CDS 3492..4352
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin complement(4394..4907)
/direction=LEFT
/note="M13 ori"
/note="M13 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 5018..5606
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature 5792..5934
/note="bom"
/note="basis of mobility region from pBR322"
CDS complement(6036..6227)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/note="rop"
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
