Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry
机构：[1]Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China大德路总院检验科大德路总院检验科广东省中医院[2]Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, 510120, China广东省中医院
Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain N-15 isotope-labeled recombinant Cys C (N-15-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and N-15-Cys C was expressed in M9 medium using N-15 as the only nitrogen source. N-15-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from N-15-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of N-15-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of N-15-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)(2+) was the parent ion of N-15-Cys C and that the secondary ions of N-15-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of N-15-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.
基金：
National Key Research and Development Program of China [2019YFF0216505]; Natural Science Foundation of Guangdong ProvinceNational Natural Science Foundation of Guangdong Province [2018A0303130124, 2020A1515010668]; Specific Research Fund for TCM Science and Technology of Guangdong Provincial Hospital of Chinese Medicine [YN2016QJ15, YN2019QL01, YN2019 MJ04]; Guangzhou Science and Technology Project [201704020213]
语种：
外文
WOS：
PubmedID：
中科院(CAS)分区：
出版当年[2020]版：
大类|4 区生物
小类|4 区生化研究方法4 区生化与分子生物学4 区生物工程与应用微生物
最新[2025]版：
大类|4 区生物学
小类|4 区生化研究方法4 区生化与分子生物学4 区生物工程与应用微生物
JCR分区：
出版当年[2019]版：
Q4BIOCHEMICAL RESEARCH METHODSQ4BIOTECHNOLOGY & APPLIED MICROBIOLOGYQ4BIOCHEMISTRY & MOLECULAR BIOLOGY
最新[2023]版：
Q4BIOCHEMICAL RESEARCH METHODSQ4BIOCHEMISTRY & MOLECULAR BIOLOGYQ4BIOTECHNOLOGY & APPLIED MICROBIOLOGY
第一作者机构：[1]Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China[2]Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, 510120, China
共同第一作者：
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推荐引用方式(GB/T 7714)：
Zhang Qiaoxuan,Cai Zhiliang,Lin Haibiao,et al.Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry[J].PROTEIN EXPRESSION AND PURIFICATION.2021,178:doi:10.1016/j.pep.2020.105785.
APA：
Zhang, Qiaoxuan,Cai, Zhiliang,Lin, Haibiao,Han, Liqiao,Yan, Jun...&Huang, Xianzhang.(2021).Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry.PROTEIN EXPRESSION AND PURIFICATION,178,
MLA：
Zhang, Qiaoxuan,et al."Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry".PROTEIN EXPRESSION AND PURIFICATION 178.(2021)
