A novel high-throughput screening method for microbial transglutaminases with high specificity toward Gln141 of human growth hormone.
PEGylation modification has been used to improve the pharmacokinetic properties of protein-based drugs. For example, PEGylated human growth hormone (hGH) has been shown to exhibit better pharmacokinetic profiles than the unmodified hGH. Unlike chemical PEGylation of hGH that is difficult to be controlled to result in homogeneity, microbial transglutaminase (mTGase) only conjugates poly(ethelene glycol) (PEG) on glutamine-40 (Q40) and glutamine-141 (Q141) of hGH, the only glutamine residues exposed. Yet, an mTGase that can selectively conjugate PEG to only 1 glutamine residue is more desirable to control the homogeneity of the product. In this study, the authors have developed a novel high-throughput assay, with which they have identified 5 mTGase mutants that are highly specific for conjugating PEG to Q141 of hGH. In this scintillation proximity assay (SPA)-based method, the authors have (1) achieved a high expression level of active mTGase, which is toxic to the living cell, directly from Escherichia coli (0.2 U/mL/OD600) by in vivo activation; (2) developed a high-throughput affinity purification method to eliminate the strong interference of cellular protein to mTGase reaction; and (3) used therapeutic protein as the substrate. This method is highly sensitive, is easily automated, and could be generally applied to screening mTGases with desired specificity targeting on different therapeutic proteins.
Zhao
Xin
X
Beijing Novo Nordisk Pharmaceuticals Sci & Tech Co. Ltd., Beijing, China. xzho@novonordisk.com
Shaw
Allan C
AC
Wang
Jianhua
J
Chang
Chih-Chuan
CC
Deng
Jianhui
J
Su
Jing
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012680:Sensitivity and Specificity; D013302:Streptomyces / Q000201:enzymology; D013379:Substrate Specificity / Q000235:genetics; D011503:Transglutaminases / Q000378:metabolism*
D001426:Bacterial Proteins; D019382:Human Growth Hormone; D011503:Transglutaminases; D005998:Glycine