Production of human pro-relaxin H2 in the yeast Pichia pastoris

Abstract

BackgroundInitially known as the reproductive hormone, relaxin was shown to possess other therapeutically useful properties that include extracellular matrix remodeling, anti-inflammatory, anti-ischemic and angiogenic effects. All these findings make relaxin a potential drug for diverse medical applications. Its precursor, pro-relaxin, is an 18 kDa protein, that shows activity in in vitro assays. Since extraction of relaxin from animal tissues raises several issues, prokaryotes and eukaryotes were both used as expression systems for recombinant relaxin production. Most productive results were obtained when using Escherichia coli as a host for human relaxin expression. However, in such host, relaxin precipitated in the form of inclusion bodies and, therefore, required several expensive recovery steps as cell lysis, refolding and reduction.ResultsTo overcome the issues related to prokaryotic expression here we report the production and purification of secreted human pro-relaxin H2 by using the methylotrophic yeast Pichia pastoris as expression host. The methanol inducible promoter AOX1 was used to drive expression of the native and histidine tagged forms of pro-relaxin H2 in dual phase fed-batch experiments on the 22 L scale. Both protein forms presented the correct structure, as determined by mass spectrometry and western blotting analyses, and demonstrated to be biologically active in immune enzymatic assays. The presence of the tag allowed to simplify pro-relaxin purification obtaining higher purity.ConclusionsThis work presents a strategy for microbial production of recombinant human pro-relaxin H2 in Pichia pastoris that allowed the obtainment of biologically active pro-hormone, with a final concentration in the fermentation broth ranging between 10 and 14 mg/L of product, as determined by densitometric analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0319-0) contains supplementary material, which is available to authorized users.

Secretion Mechanism across Wall

“…Trx-HPV16-L2 immunogen alpha-factor signal peptide Vaccine [84] Horseradish peroxidase MATα prepro secretion signal [85] Candida antartica lipase Human Pro-relaxin L2 alpha-factor signal peptide Therapeutic applications [86] FSL2, Lipase S. cerevisiae α-factor signal sequence…”

Seleno-relaxin analogues: effect of internal and external diselenide bonds on the foldability and a fibrosis-related factor of endometriotic stromal cells

“…After oxidative folding, C-peptide is removed by protease to obtain the mature relaxin. [27][28][29][30] However, synthesis of novel relaxins using this method requires the development of corresponding synthetic genes and thus is not suitable for the rapid production of a wide variety of formulations. Alternatively, chemical synthetic technology facilitates the introduction of artificial amino acids and chemical modifications of proteins.…”

Selymatra: A web application for protein‐profiling analysis of mass spectra

“…In particular, a large amount of data must be accumulated in order to identify putative biomarkers, and often pre-treatment steps of samples become necessary to enhance differences and reduce the background noise in the MS spectra. In addition, several other techniques are later necessary for the identification of biomarkers and protein-protein interactions, such as sequencing by MSMS-QTOF, immuno-precipitation and western blotting [9,[15][16][17][18]. These limits made it necessary to develop new strategies in order to improve these methodologies.…”

“…In addition, several other techniques are later necessary for the identification of biomarkers and protein-protein interactions, such as sequencing by tandem quadrupole-time of flight mass spectrometry (MSMS-QTOF), immunoprecipitation, and Western blotting. 9,[15][16][17][18] These limits made it necessary to develop new strategies in order to improve these methodologies.…”