An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase.
A chimera between gene segments of Protein A and a mutated alkaline phosphatase (lysine328 mutated to alanine) of Escherichia coli has been constructed. This chimeric gene was cloned in a T7 promoter-based IPTG-inducible expression vector. The chimeric protein was expressed in E. coli and was efficiently secreted into the periplasm, from which it could be easily purified by a combination of ion-exchange and gel permeation chromatography. The purified chimera was found to be thermostable and exhibited both IgG binding and high alkaline phosphatase activity. It was used as a probe in enzyme-linked immunosorbent assays and results indicate that it is a promising substitute for secondary antibodies in enzyme-linked immunoassays.
Chowdhury
P S
PS
Department of Biochemistry, University of Delhi South Campus, India.
Kushwaha
A
A
Abrol
S
S
Chaudhary
V K
VK
D000469:Alkaline Phosphatase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000469:Alkaline Phosphatase