Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris

Abstract

Background: 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. 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.

Results: In 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.

Conclusions: Our 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.

Keywords: Furin; Furin cleavage site; Kex2; Pichia pastoris; Proprotein convertase; YPS1.

Fig. 1

Cleavage of furin cleavage site in Pichia pastoris. a Schematic diagram of expression vectors using in this study. b Western blot of expressed products using anti-GFP antibody, the culture supernatants didn’t be treated with Endo Hf endoglycosidase which cleaves the chitobiose core of high mannose from N-linked glycoprotein. c Western blot of expressed products using anti-GFP antibody, the culture supernatants were treated with Endo Hf to show α-factor-GFP fusion protein after high mannose chains were removed from α-factor peptide. d Quantitative analysis of the western blot results, compared GFP with GFP and α-factor-GFP

Fig. 2

Arg residue at position P1 or P4 in furin cleavage sites effects on cleavage efficiency in Pichia pastoris. a Schematic diagram of expression vectors mutated furin cleavage sites from Arg to Ile at P1 or P4 position. b–d Western blot results of expressed products with three different furin cleavage sites and their corresponding mutants. The western blot samples were treated with Endo Hf. e–g Quantitative analysis of the western blot results. **p < 0.01, ***p < 0.001

Fig. 3

YPS1 doesn’t involve in processing the furin cleavage site in Pichia pastoris. a Schematic diagram for constructing a disruption vector with left homology arm and right homology arm of YPS1 gene. b Schematic diagram for knockout of YPS1 gene by homologous recombination. Zeocin can be used for selection of YPS1 knockout strains. Two pairs primers, as shown in figure, were designed to confirm the disrupted strains. c Genomic PCR of YSP1 disrupted and wild type strains using two pairs primers (YI F, YI R and YO F, AOX1 R). A band can be amplified by YI F and YI R primers only if YPS1 gene doesn’t be deleted, and a band can be amplified by YO F and AOX1 primers if YPS1 gene is successfully removed by the disruption construct. d Western blot results of wild type strains. The western blot samples were treated with Endo Hf. e Western blot results of YPS1 disruption strains. The western blot samples were treated with Endo Hf. f Quantitative analysis of the western blot results. NS not statistically significant

Fig. 4

Kex2 involved in processing the furin cleavage site in Pichia pastoris. a Schematic diagram for constructing a targeting vector with left homology arm and right homology arm of Kex2 gene. b Schematic diagram for knockout of Kex2 gene by homologous recombination. Zeocin can be used for selection of Kex2 disruption strains. Two pairs primers, as shown in figure, were designed to confirm the disruption strains. c Genomic PCR of Kex2 disruption and wild type strains using two pairs primers (YI F, YI R and YO F, AOX1 R). A band can be amplified by YI F and YI R primers only if Kex2 gene doesn’t be deleted, and a band can be amplified by YO F and AOX1 primers if Kex2 gene is successfully removed by the targeting construct. d Western blot results of wild type strains. The western blot samples were treated with Endo Hf. e Western blot results of Kex2 disruption strains. The western blot samples were treated with Endo Hf. f Quantitative analysis of the western blot results. ***p < 0.001

Fig. 5

Heterologous expression of furin increased cleavage efficiency of furin cleavage sites in Pichia pastoris. a Genomic PCR for analyzing integration of furin gene into GS115 genome using AOX1 general primers. b Expression analysis of integrated furin gene in Pichia pastoris GS115 by RT-PCR with β-actin and furin specific primers. c Western blot results of GS115 strains. The western blot samples were treated with Endo Hf. d Western blot results of GS115-furin strains. The western blot samples were treated with Endo Hf. e Quantitative western blot analysis of cleavage efficiency of three furin cleavage sites. *p < 0.05, ***p < 0.001

Fig. 6

Overexpression of Kex2 increased cleavage efficiency of furin cleavage sites in Pichia pastoris. a Genomic PCR for analyzing integration of Kex2 gene into GS115 genome using 5′ GAP general upstream and Kex2 specific downstream primers. b Kex2 overexpression analysis in GS115-Kex2 strains by RT-PCR. Expression of β-actin was used as control. c Western blot results of GS115 strains. The western blot samples were treated with Endo Hf. d Western blot results of GS115-Kex2 strains. The western blot samples were treated with Endo Hf. e Quantitative western blot analysis of cleavage efficiency of three furin cleavage sites in GS115 and GS115-Kex2 strains. *p < 0.05, ***p < 0.001