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pMalc2x載體質粒圖譜和多克隆位點信息

pMalc2x載體簡介

pMAL?系統是一種高效的蛋白融合表達及純化系統。pMAL載體含有編碼麥芽糖結合蛋白（Maltose Binding Protein, MBP）的大腸桿菌malE基因，其下游的多克隆位點便于目的基因插入，表達N端帶有MBP的融合蛋白。通過"tac"強啟動子和malE翻譯起始信號使克隆基因獲得高效表達，并進一步利用MBP對麥芽糖的親和性達到用Amylose柱對融合蛋白的一步親和純化。

The system uses the pMAL vectors which are designed so that insertion interrupts a lacZα gene allowing a blue-to-white screen for inserts on X-gal (5). pMAL-c2 series has an exact deletion of the malE signal sequence, resulting in cytoplasmic expression of the fusion protein. pMAL-p2 series contains the normal malE signal sequence, which directs the fusion protein through the cytoplasmic membrane. pMAL-p2 fusion proteins capable of being exported can be purified from the periplasm. Between the malE sequence and the polylinker there is a spacer sequence coding for 10 asparagine residues. This spacer insulates MBP from the protein of interest, increasing the chances that a particular fusion will bind tightly to the amylose resin. The vectors also include a sequence coding for the recognition site of a specific protease. This allows the protein of interest to be cleaved from MBP after purification, without adding any vector-derived residues to the protein (6). For this purpose, the polylinker includes a restriction site superimposed on the sequence coding for the site of the specific protease. This is where the gene of interest is inserted. An EcoR I site in the same reading frame as that of λgt11 and a number of other useful sites are present directly downstream. The vectors also include the M13 origin of DNA replication which allows the production of single-stranded DNA for sequencing and mutagenesis by infecting with M13KO7 helper phage.