Comparative characterization of recombinant ZZ protein–alkaline phosphatase and its application in enzyme immunoassays

• Published in: Applied microbiology and biotechnology 2013, Vol.97 (1), p.153-158
• Main Authors: Tang, Jin-Bao, Yang, Hong-Ming, Liang, Shu-Juan, Chen, Yong, Mu, Qing-Jie, Zhang, Jin-Bao
• Format: Article
• Language: English
• Subjects: Alkaline phosphatase; Alkaline Phosphatase - genetics; Antibodies; Antibody Affinity; Biomedical and Life Sciences; Biotechnological Products and Process Engineering; Biotechnology; Catalytic activity; Conjugates; E coli; Enzymatic activity; Enzyme-linked immunosorbent assay; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fusion protein; Immunoassay; Immunoassays; Immunoenzyme Techniques - methods; Immunoglobulin G; Immunoglobulin G - genetics; Life Sciences; Microbial Genetics and Genomics; Microbiological research; Microbiology; Microorganisms; Phosphatase; Phosphatases; Physiological aspects; Proteins; Recombinant Fusion Proteins - genetics; Recombinant proteins; Sensitivity and Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-4303-x

A functional fusion protein, which consists of an antibody and an enzyme that can be used in enzyme immunoassays, has been constructed. However, a quantitative comparison of the characteristics of fusion proteins and chemical conjugates of the parents, which are functionally produced in a uniform microbial system, has not been adequately achieved. In this study, a fusion protein between the ZZ protein and Escherichia coli alkaline phosphatase (AP) and the parental ZZ protein and AP for chemical conjugate was functionally produced in the same bacterial system. A detailed examination of the ZZ–AP fusion protein and the effect of the ZZ–AP chemical conjugate on IgG affinity and enzymatic activity were performed. Compared with the parents, the equilibrium dissociation constant of ZZ–AP conjugate decreased by 32 % and catalytic activity decreased by 24 %, whereas the ZZ–AP fusion retained full parental activities and exhibited an approximately tenfold higher sensitivity than that of ZZ–AP conjugate in enzyme-linked immunosorbent assay. Thus, ZZ–AP fusion is a promising immunoreagent for IgG detection and a potential biolinker between antibodies and reporter enzymes (i.e., IgG–ZZ–AP fusion complex) in immunoassays.