- Open Access
Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris
© The Author(s) 2018
- Received: 29 August 2018
- Accepted: 31 October 2018
- Published: 8 November 2018
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.
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.
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.
- Pichia pastoris
- Furin cleavage site
- Proprotein convertase
Many important peptides or proteins are synthesized intracellularly as higher-molecular-mass inactive precursors consisted of signal peptide, propeptide and mature peptide in eukaryotes from yeasts to mammals [1, 2]. Release of propeptide from inactive precursor proteins are proteolyzed by proprotein convertases within the secretory pathway, leading to the production of biologically active proteins or peptides. The product of the KEX2 gene, Kex2 [also known as kexin (EC 184.108.40.206)], was the first identified proprotein convertase involved in the processing of α-mating factor and killer toxin precursors in Saccharomyces cerevisiae. Kex2 belonging to the subtilisin family has a catalytic domain similar to bacterial serine proteases and cleaves its precursors at paired basic residues . Proprotein convertase furin, encoded by the fur gene, is the homologue of Kex2 in mammal . Furin is expressed as a 794 amino acid precursor and rapidly converted into active mature form by two intramolecular autocatalytic cleavage (the first cleavage site, Arg-Ala-Lys-Arg107; the second cleavage site, Arg-Gly-Val-Thr-Lys-Arg75) . The structure of furin includes a signal peptide, a propeptide, a subtilisin-like catalytic domain, a Homo B domain, a Cys-rich domain, a transmembrane domain and a cytoplasmic domain. Furin is capable of processing a wide variety of precursors including growth factors, hormones, neuropeptides, transcription factors, plasma proteins, receptors, viral envelope proteins, matrix metalloproteinases and bacterial exotoxins [1, 6]. Many substrates of furin have important physiological functions and possess great clinical values. It is attractive for the pharmaceutical industry to produce these substrates via recombinant protein expression systems.
Pichia pastoris, reclassified as Komagataella phaffi , has become a successful host organism for production of recombinant proteins due to the benefits of high cell density cultivation, the simplicity of genetic manipulation, growth on inexpensive media, efficient secretory capabilities with a low level of endogenous protein secretion, the strong and tightly inducible AOX1 promoter and the ability of post-translational modifications to proteins [8, 9]. Currently, over 1000 proteins have been produced in P. pastoris . In our previous studies, we heterologously expressed two furin substrates using the DNA sequences encoding the mature peptides in P. pastoris. But the recombinant proteins couldn’t form the proper native structures [11, 12]. Several studies have found that the propeptides in some precursors are important for the production of active mature peptides. The propeptides can act as intramolecular chaperones to assist the proper folding of mature peptides such as activin A and transforming growth factor-beta 1 (TGF-β1) , bone morphogenetic protein-4 (BMP-4)  and cathepsin L , or stabilize the mature peptides to increase the their transport from ER to Golgi [16, 17]. Therefore, co-expression of propeptide and mature peptide sequence may provide a strategy for efficient production of biologically active proteins processed by furin in P. pastoris. 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 DNA sequences of furin substrates containing the propeptides and mature peptides.
In this study, we used reporter proteins containing furin cleavage sites between the propeptide and mature peptide to systematically evaluate the ability of P. pastoris GS115 on processing furin cleavage sites. Our study will shed light on how to improve the efficiencies of producing biological active furin substrates in P. pastoris.
Furin cleavage sites can be cleaved in Pichia pastoris
At P1 position, both furin and Saccharomyces cerevisiae Kex2 exhibit extremely stringent specificity for Arg. Studies on furin substrate specificity have also shown that replacement of P1 Arg results in undetectable cleavage . Substitution with similar positive charge amino acid, such as Lys or ornithine, at P1 position in Saccharomyces cerevisiae Kex2 peptidyl-MCA substrates also resulted in an approximately 100-fold drop in kcat/KM relative to the canonical substrate sequence . The cleavage efficiencies of all three furin processing sites were also significantly decreased when Arg residue at position P1 was mutated into Ile. For Arg-Ser-Ile-Arg motif, the cleavage was completely abolished. For Arg-Ser-Arg-Arg and Arg-Ser-Lys-Arg motifs, the cleavage efficiencies were greatly decreased. Our results showed that Arg residue at P1 is essential for cleavage in P. pastoris. For Saccharomyces cerevisiae Kex2, most of its selectivity towards substrates arises through interactions at position P1 and P2 [25, 26]. In P. pastoris, the amino acid at P2 seems to be less important if there is Arg at P4, suggesting that the selectivity of protease to substrates in P. pastoris is more like furin which generates most of its selectivity through interactions of Arg at P1 and P4.
Kex2, but not YSP1, is responsible for processing the furin cleavage site in Pichia pastoris
Heterologous expression of furin or overexpression of Kex2 significantly increased cleavage efficiency of furin cleavage sites in Pichia pastoris
Recently, studies on ligase derived from fungus showed that co-expression of propeptide and mature peptide in P. pastoris helped mature peptide form the proper protein conformation [32, 33]. A large number of propeptide of precursor proteins have been identified as intramolecular chaperones (IMCs) and assist the proper folding of mature peptides. Propeptides of precursors processed by furin were also found to be critical in correct protein folding of mature peptides [13, 14]. Further researches are needed to verify whether the expression of the furin substrate DNA sequence consisted of both the propeptide and mature peptide in P. pastoris help enhance to activity of the mature peptide. If this strategy proves to produce more active furin substates in comparison with direct expression of the mature peptide DNA sequence, our GS115-furin and GS115-Kex2 strains may be good choices for expressing precursors processed by furin in P. pastoris.
In this study we showed that Kex2, but not YPS1, in P. pastoris is capable of processing furin cleavage sites and the Arg residue at position P1 or P4 of furin cleavage sites significantly affect the cleavage efficiency. Overexpression of both furin or Kex2 in P. pastoris effectively increased the processing efficiency of furin cleavage sites, providing an attractive direction for efficiently express bioactive furin substrates in P. pastoris.
Enzymes and reagents
Restriction enzymes, T4 DNA ligase, Taq DNA polymerase, polymerase chain reaction (PCR) reagent, Prime STAR polymerase, and PCR reagent were obtained from Takara Bio Inc. The Endo Hf was purchased from New England Biolabs, Inc. SDS-PAGE Protein Marker was supplied by Thermo Fisher Scientific. Primers were all synthesized at Sangon Biotech (China). PCR Purification Kit was from Promega Corporation. Plasmid Mini Kit and Point mutation kit were from TransGen Biotech (China). All other chemicals used were acquired from Sigma Aldrich Chemicals P Ltd. and Sinopharm Chemical Reagent (China).
Strains and plasmids
Escherichia coli Top10F’ was used as the cloning host. P. pastoris GS115(his 4−) was used as the expression host. Plasmid pPIC9 and pGAPZB were used as gene expression vectors. All of them were purchased from Thermo Fisher Scientific (Invitrogen). The recombinant plasmid pPIC9-GFP with different furin cleavage sites, YPS1 disruption vector pYPS1Δ, Kex2 disruption vector pKex2Δ, pGAPZB-Kex2 and pGAPZB-furin were constructed and stored in our lab.
Preparation of DNA constructs
Primers using in this study
Primer sequence (5′–3′)
Preparation for expression vectors
Preparation for disruption vectors
Left arm F
Left arm R
Right arm F
Right arm R
Left arm F
Left arm R
Right arm F
Right arm R
Identification for disrupted strains
Identification for GS115-furin and GS115-Kex2 strains
To construct YPS1 and Kex2 gene disruption vectors, The P. pastoris GS115 genome was extracted and used as template to amplify the left homology arm and right homology arm with two pairs of specific primers to YPS1 or Kex2 gene. The left arm fragment contains PstI site at 5′ end and XhoI at 3′ end, and the right arm fragment contains BglII site at 5′ end and PstI at 3′ end. pPICZα A was used as the backbone to generate the disruption vectors. Both of pPICZα A and the left arm fragment were digested with the PstI and XhoI restriction endonucleases and ligated to produce an intermediate plasmid, The intermediate plasmid then was digested with the PstI and BglII restriction endonucleases. The right arm fragment digested with the same restriction endonucleases was inserted into the intermediate plasmid at the PstI and BglII sites to produce the disruption vector.
In order to overexpress Kex2 protease in P. pastoris GS115, Kex2 gene was amplified from P. pastoris GS115 genome by PCR and cloned into pGAPZ B expression vector at EcoRI and XhoI sites. To heterologous expression of furin proprotein convertase in GS115, furin cDNA was amplified by PCR using pGEM-furin plasmid (a gift kindly provided by professor Gary Thomas from Department of Cell and Developmental Biology, Oregon Health and Science University, School of Medicine, Portland, USA) as the template, and cloned into pGAPZ B expression vector at EcoRI and XhoI sites.
The recombinant plasmids pPIC9-GFP(RSIR), pPIC9-GFP(RSRR), pPIC9-GFP(RSKR), pPIC9-GFP(ISIR), pPIC9-GFP(RSII), pPIC9-GFP(ISRR), pPIC9-GFP(RSRI), pPIC9-GFP(ISKR) and pPIC9-GFP(RSKI) were linearized with SalI restriction endonuclease. pKex2Δ, pYPS1Δ were linearized with PstI restriction endonuclease. pGAPZ B-Kex2 and pGAPZ B-furin were linearized with AvrII restriction endonuclease. 10 μg linearized plasmid DNA was transformed into P. pastoris GS115 competent cells by electroporation (1.5 kV, 25 µF, 200 Ω) using a Bio-Rad Gene Pulser (Hercules, USA). The transformed cells were screened on MD (0.34% YNB, 4 × 10–5% biotin, 1% dextrose, and 1.5% agar) or YPD (1% yeast extract, 2% peptone, 2% dextrose and 1.5% agar) in the presence of 25 µg/ml Zeocin (Invitrogen) plates.
Expression of protein in shaking flasks
Colonies were picked into BMGY medium (1% yeast extract, 2% peptone, 0.34% YNB, 4 × 10−5 % biotin, 0.5% methanol, 100 mM potassium phosphate, pH 6.0,) and incubated in an orbital shaker at 220 rpm for 24 h until the OD600 nm reached 2–5. The supernatant was discarded by centrifugation at 1500 rcf for 10 min at room temperature, and the cells were resuspended in 10 ml BMMY medium (1% yeast extract; 2% peptone; 100 mM phosphate buffer, pH 6.0; 1.34% yeast nitrogen base, 4 × 10−5 % biotin; and 1% methanol) to a final OD600 nm = 1 in 250 ml shaking flasks. Methanol was added to induce expression of GFP every 24 h. After shaking in flasks for 72 h, the supernatant was harvested.
Deglycosylation by Endo Hf
Due to the presence of glycosylation sites in the α-factor peptide, the expressed α-factor-GFP fusion protein was glycosylated. Endo Hf (New England Biolabs) was used to remove N-glycan of α-factor-GFP fusion protein. Briefly, the culture supernatant was boiled for 10 min in denatured buffer containing 0.4 M DTT and 0.5% SDS, and deglycosylation was then performed by treatment with Endo Hf at 37 °C for overnight according to the instructions of the manufacture. The buffer employed in these enzyme reactions was 50 mM sodium citrate (pH 5.5) for Endo Hf.
Determination of the cleavage efficiency of furin cleavage sites in Pichia pastoris by western blotting
Cleavage efficiency to different furin cleavage sites in P. pastoris was validated by western blotting. The proteins in culture supernatant were separated by SDS-PAGE (12% gel), and then transferred onto a Protran nitrocellulose membrane (Signa Amersham) using a Mini Trans-Blot Cell (Bio-Rad). The membranes were blocked with 5% fat-free milk and incubated overnight at 4 °C with anti-GFP antibodies at 1:500 dilutions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). IRDye 800CW goat anti-mouse immunoglobulin G (LI-COR Biosciences) was used as the secondary antibody at 1:1000 dilutions. Detection and quantification of proteins were performed using Odyssey imaging systems (LI-COR Biosciences). Cleavage efficiency was calculated by the following equation: cleavage efficiency = sum of cleaved GFP band intensities/sum of cleaved GFP and α-factor-GFP fusion protein band intensities. All the experiments were repeated at least three times independently.
Total RNA of Kex2 or furin knockin GS115 strains was obtained using Yeast RNAiso Kit (Takara Bio Inc.) and cDNA was synthesized from 500 ng of the total RNA using Reverse Transcription System (Takara Bio Inc.). The primer sequences used in this work are shown in Table 1. The PCR mixture was first heated to 95 °C for 5 min, then entering 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. The PCR products were analyzed by 1.5% agarose gel electrophoresis with ethidium bromide staining.
YH and YLi conceived and designed the experiments. YLo performed the majority of the laboratory work. SL and TL mutated furin cleavage sites. JW and YZ constructed plasmids. YH wrote the manuscript. YLi revised the manuscript. All authors read and approved the final manuscript.
The authors would especially like to thank Prof. Gary Thomas for the kind gift of pGEM-furin plasmid.
The authors declare that they have no competing interests.
Availability of data and materials
The data supporting the conclusions of this article are included with the article.
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This project is financed by the Natural Science Foundation of Fujian Province (2016J01146) and the Scientific Research Innovation Team Construction Program of Fujian Normal University (IRTL1702).
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