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

BACKGROUND: Initially 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. RESULTS: To 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. CONCLUSIONS: This 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.

Production and purification of higher molecular weight chondroitin by metabolically engineered Escherichia coli K4 strains.

The capsular polysaccharide obtained from Escherichia coli K4 is a glycosaminoglycan-like molecule, similar to chondroitin sulphate, that has established applications in the biomedical field. Recent efforts focused on the development of strategies to increase K4 polysaccharide fermentation titers up to technologically attractive levels, but an aspect that has not been investigated so far, is how changes in the molecular machinery that produces this biopolymer affect its molecular weight. In this work, we took advantage of recombinant E. coli K4 strains that overproduce capsular polysaccharide, to study whether the inferred pathway modifications also influenced the size of the produced polymer. Fed-batch fermentations were performed up to the 22 L scale, in potentially industrially applicable conditions, and a purification protocol that allows in particular the recovery of high molecular weight unsulphated chondroitin, was developed next. This approach allowed to determine the molecular weight of the purified polysaccharide, demonstrating that kfoF overexpression increased polymer size up to 133 kDa. Higher polysaccharide titers and size were also correlated to increased concentrations of UDP-GlcA and decreased concentrations of UDP-GalNAc during growth. These results are interesting also in view of novel potential applications of higher molecular weight chondroitin and chondroitin sulphate in the biomedical field.