title: High-level expression of recombinant protein A in Escherichia coli.
abstract: 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.
authors.affiliation: Laboratory of Protein and Peptide Chemistry, Institute of Basic Medical Sciences, Beijing, China.
authors.forename: S
authors.initials: S
authors.lastname: Cai
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: B
authors.initials: B
authors.lastname: Qiang
authors.forename: Z
authors.initials: Z
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A