Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficien...

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Bibliographic Details
Published in:Amino acids 2015-06, Vol.47 (6), p.1117-1125
Main Authors: Guo, Yu-Qi, Wu, Qing-Ping, Shao, Xiao-Xia, Shen, Ting, Liu, Ya-Li, Xu, Zeng-Guang, Guo, Zhan-Yun
Format: Article
Language:English
Subjects:
Analytical Chemistry
Biochemical Engineering
Biochemistry
Biomedical and Life Sciences
Humans
Insulin - biosynthesis
Insulin - chemistry
Insulin - genetics
Isotope Labeling - methods
Life Sciences
Neurobiology
Original Article
Peptide Biosynthesis
Peptides - chemistry
Peptides - genetics
Peptides - metabolism
Pichia - genetics
Pichia - metabolism
Proteins - chemistry
Proteins - genetics
Proteomics
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - genetics
Relaxin - biosynthesis
Relaxin - chemistry
Relaxin - genetics
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Summary:Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly 15 N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified 15 N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.
ISSN:0939-4451
1438-2199
DOI:10.1007/s00726-015-1939-8