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 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.
Institute of Protein Research, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
Yu-Qi
YQ
Guo
Qing-Ping
QP
Wu
Xiao-Xia
XX
Shao
Ting
T
Shen
Ya-Li
YL
Liu
Zeng-Guang
ZG
Xu
Zhan-Yun
ZY
Guo
D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012065:Relaxin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D007328:Insulin; C084797:Leydig insulin-like protein; D010455:Peptides; D011506:Proteins; C480238:RLN3 protein, human; D011993:Recombinant Fusion Proteins; D012065:Relaxin