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Updated: Jul 2, 2026

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
Published on: December 9, 2017
Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis. Cooperative
H Leitolf1, K P Tong, M Grossmann
1Laboratory of Molecular Endocrinology, Division of Endocrinology, Diabetes, and Nutrition, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Abstract:
We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the alphaL3 loop of the common alpha-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent alphaL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the alphaL3 loop analog compared with both the alphaL1 loop analog and the hTSH-betaL3 loop analog was observed. We demonstrated that spatially distinct regions of the common alpha-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.
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