Title
Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli
Author
Jin-Hee Lee, Ji-Seon Jeong, Sook-Kyung Kim, Jimyeong Song, Ji Youn Lee, Soyun Baek, Jun-Hyuk Choi
Year
2016
Journal
Journal of Chromatography B
Abstract
Isotopically labeled proteins have been used as internal standards for mass spectrometry (MS)-based absolute protein quantification. Although this approach can provide highly accurate analyses of proteins of interest within a complex mixture, one of the major limitations of this method is the difficulty in preparing uniformly labeled standards. Human growth hormone (hGH) is one of the most important hormones that circulate throughout the body, and its measurement is primarily of interest in the diagnosis and treatment of growth disorders. In order to provide a useful internal standard for MS-based hGH measurement, we describe an efficient strategy to produce a potentially valuable, stable isotope-labeled hGH with high purity and yield. The strategy involves the following steps: solubilization of hGH under labeling conditions, detection of stable isotope incorporation, large-scale purification, analysis of the labeled protein, and assessment of the labeling efficiency. We show that the yield of soluble hGH under selective isotopic labeling conditions can be greatly increased by optimizing protein expression and extraction. Our efficient method for generating isotopically labeled hGH does not influence the structural integrity of hGH. Finally, we assessed the efficiency of stable isotope labeling at the intact protein level, and the result was further verified by amino acid analysis. These results clearly indicate that our labeling approach allows an almost complete incorporation of 13C615N4-arginine into the hGH expressed in E.coliwithout detectable isotope scrambling.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry