Abstract
BackgroundProprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides.ResultsIn this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites.ConclusionsOur investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.
Highlights
Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal
Furin cleavage sites can be cleaved in Pichia pastoris In our previous study, we examined all reported precursors processed by furin and found there were three main cleavage sites between propeptides and mature peptides: Arg-Xaa-Lys-Arg (41%), Arg-Xaa-Arg-Arg (31.5%) and Arg-Xaa-Xaa-Arg (11%) [18]
In order to determine if mammalian furin cleavage sites can be cleaved in P. pastoris, three reporter vectors containing different furin cleavage sites (Arg-SerLys-Arg, Arg-Ser-Arg-Arg or Arg-Ser-Ile-Arg) between α-factor propeptide and GFP were constructed (Fig. 1a)
Summary
Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications It is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides. Coexpression of propeptide and mature peptide sequence may provide a strategy for efficient production of biologically active proteins processed by furin in P. pastoris It is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the DNA sequences of furin substrates containing the propeptides and mature peptides
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