载体编号：P0056
载体名称：pMAL-C2X
载体抗性：Amp
载体大小：约6.7Kbp
载体宿主：E.coli
载体特点：The pMAL™-2 vectors 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. The method uses the strong “tac” promoter and the malE translation initiation signals to give high-level expression of the cloned sequences, and a one-step purification of the fusion protein using MBP’s affinity for maltose. 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 or JM107. 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. 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-2 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-c2X and
pMAL-p2X vectors that are included in the system encode the site for Factor Xa. 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. The vectors pMAL-c2G (NEB #N8068) and pMAL-p2G (NEB #N8069) encode the site for Genenase™I (NEB #P8075), which cleaves following the sequence His-Tyr. The vectors pMAL-c2E (NEB #N8066) and pMAL-p2E (NEB #N8067) encode the site for Enterokinase (NEB #P8070), which cleaves following the sequence Asp-Asp-Asp-Asp-Lys. In the large majority of cases, fusion protein expressed from a pMAL-c2 plasmid constitutes 20–40% of the total cellular protein, while fusion protein expressed from a pMAL-p2 plasmid constitutes 1–5% of the total cellular protein. For pMAL-c2 vectors, a band corresponding to the fusion protein can usually be seen by running a small sample of induced cells on an SDSPAGE gel. The yield of fusion protein from the affinity purification ranges up to 200 mg/liter culture, with typical yields in the range of 10–40 mg/liter. The yield varies greatly depending upon the sequences fused to malE. In cases where the yield has been compared directly, the pMAL-c2 vectors (no signal sequence) give approximately 10-fold more protein in the affinity purification than pMAL-p2 vectors. The pMAL-p2 vectors are useful for cases where export to the periplasm is desirable, e.g. for purification or disulfide bond formation. 75% of the fusions made so far have worked in the affinity purification. In the cases that have not worked, the fusion binds to the column poorly or not at all, is degraded by E. coli proteases, or is insoluble. The pMAL-2 vectors also contain the origin of DNA replication of the E. coli bacteriophage M13, which allows the production of single-stranded DNA by infection of cells bearing a pMAL-2 plasmid with a helper phage. The single-stranded plasmid DNA can be used for sequencing using a primer available from NEB (see Sequencing, page 9), or for oligonucleotidedirected mutagenesis.
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