Cai S, Liu Y, Qiang B, Yao Z
Laboratory of Protein and Peptide Chemistry, Institute of Basic Medical Sciences, Beijing, China.
Chin J Biotechnol. 1992;8(2):93-8.
A high-level expression plasmid pPA-3 was constructed, which yielded up to 20% of soluble cell proteins as recombinant Protein A in E. coli DH5 alpha strain by heat shock induction. The recombinant Protein A contained only the five domains of native Protein A that bind with the Fc part of IgG. The molecular weights of the recombinant Protein A expressed in E. coli were determined to be 33kDa, 32.2kDa, 29.5kDa and 28.6kDa by SDS-PAGE and Western-blotting. The diversity of the molecular weights may be due to proteolysis, and a possible cleavage site is proposed. Some of the protein was purified with coupled human IgG by one-step affinity chromatography. The reactivity of the protein was compared with that of native Protein A and found that this protein could bind with more IgG than native Protein A in equal quantities of proteins.
构建了一个高水平表达质粒pPA - 3，通过热休克诱导，在大肠杆菌DH5α菌株中，该质粒产生的重组蛋白A高达可溶性细胞蛋白的20%。重组蛋白A仅包含天然蛋白A与IgG的Fc部分结合的五个结构域。通过SDS - PAGE和蛋白质印迹法测定，在大肠杆菌中表达的重组蛋白A的分子量分别为33kDa、32.2kDa、29.5kDa和28.6kDa。分子量的差异可能是由于蛋白水解作用，并提出了一个可能的切割位点。部分蛋白通过一步亲和层析法与偶联的人IgG进行纯化。将该蛋白的反应性与天然蛋白A的反应性进行比较，发现在等量蛋白质中，该蛋白比天然蛋白A能结合更多的IgG。
