A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-beta-galactosidase.
The lacG gene encoding the 6-phospho-beta-galactosidase (E.C.3.2.1.85) of Staphylococcus aureus was fused to the protein A gene in the plasmid pRIT2T. Escherichia coli cells containing this plasmid produce a fusion protein with both IgG binding and 6-phospho-beta-galactosidase activities after heat induction. The recombinant gene was overexpressed and the hybrid protein was purified to homogeneity in high yield. The chimeric protein was shown to have almost identical enzymatic characteristics to pure 6-phospho-beta-galactosidase. This result leads to the conclusion that a free N-terminus of the 6-phospho-beta-galactosidase is not required for biological activity. The hybrid protein of protein A and 6-phospho-beta-galactosidase was used as an enzyme conjugate in enzyme-linked immunosorbent assays (ELISA). The experiments presented demonstrate that the 6-phospho-beta-galactosidase is a suitable fusion partner in various diagnostic applications where an unique biological activity is required.
Witt
E
E
Abteilung für Biologie, Ruhr-Universität Bochum, Germany.
Stewart
G C
GC
Hengstenberg
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C036210:immunoglobulin G-sepharose; D012685:Sepharose; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase