Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli.
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 of13C615N4-arginine into the hGH expressed in E.coli without detectable isotope scrambling.
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea; Department of Bio-Analytical Science, University of Science & Technology, 217 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Jin-Hee
JH
Lee
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea; Department of Bio-Analytical Science, University of Science & Technology, 217 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Ji-Seon
JS
Jeong
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Sook-Kyung
SK
Kim
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea; Department of Bio-Analytical Science, University of Science & Technology, 217 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Jimyeong
J
Song
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Ji Youn
JY
Lee
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea; Department of Bio-Analytical Science, University of Science & Technology, 217 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea.
Soyun
S
Baek
Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea; Department of Bio-Analytical Science, University of Science & Technology, 217 Gajeong-ro, Youseong-gu, Daejeon 34113, Republic of Korea, Republic of Korea. Electronic address: junchoi@kriss.re.kr.
Jun-Hyuk
JH
Choi
D004926:Escherichia coli / Q000235:genetics; D019382:Human Growth Hormone / Q000235:genetics*; D007553:Isotope Labeling; D011994:Recombinant Proteins / Q000235:genetics
D011994:Recombinant Proteins; D019382:Human Growth Hormone
Escherichia coli; Human growth hormone; Purification; Recombinant protein; Stable isotope