title: Production of human pro-relaxin H2 in the yeast Pichia pastoris.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy. donatella.cimini@unina2.it.
authors.forename: D
authors.initials: D
authors.lastname: Cimini
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: K Della
authors.initials: KD
authors.lastname: Corte
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Finamore
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Andreozzi
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Stellavato
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: A V A
authors.initials: AV
authors.lastname: Pirozzi
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Ferrara
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Formisano
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy.
authors.forename: M
authors.initials: M
authors.lastname: De Rosa
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia I, 80126, Naples, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Chino
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia I, 80126, Naples, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Lista
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia I, 80126, Naples, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Lombardi
authors.affiliation: Department of Chemical Sciences, University of Naples Federico II, Via Cintia I, 80126, Naples, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Pavone
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples and University of Campania Luigi Vanvitelli, via de Crecchio 7, 80138, Naples, Italy. chiara.schiraldi@unina2.it.
authors.forename: C
authors.initials: C
authors.lastname: Schiraldi
mesh_terms: D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012065:Relaxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: C095068:RLN2 protein, human; D011994:Recombinant Proteins; D012065:Relaxin
keywords: IMAC; LCMS; Pichia pastoris; Pro-hormone; Pro-relaxin; Protein instability