Isolation of <i>Pichia pastoris PIR</i> genes and their utilization for cell surface display and recombinant protein secretion

Khasa, Yogender Pal; Conrad, Sara K.; Şengül, Mustafa; Plautz, Sarah A.; Meagher, Michael M.; İnan, Mehmet

doi:10.1002/yea.1832

Cited by 35 publications

(13 citation statements)

References 49 publications

(50 reference statements)

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“…5, lanes 5 and 6. We sequenced the N-terminal 12 amino acids of the 30 kDa protein band and found the PIR gene family of proteins [11] having four concatameric repeats of amino acids "DDDK". This protein is found to be unique to P. pastoris when analyzed using the protein databank.…”

Section: Discussionmentioning

confidence: 99%

A New Signal Sequence for Recombinant Protein Secretion in Pichia pastoris

Govindappa¹,

Hanumanthappa²,

Krishna³

et al. 2014

Journal of Microbiology and Biotechnology

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Pichia pastoris is one of the most widely used expression systems for the secretory expression of recombinant proteins. The secretory expression in P. pastoris usually makes use of the prepro MATα sequence from Saccharomyces cerevisiae, which has a dibasic amino acid cleavage site at the end of the signal sequence. This is efficiently processed by Kex2 protease, resulting in the secretion of high levels of proteins to the medium. However, the proteins that are having the internal accessible dibasic amino acids such as KR and RR in the coding region cannot be expressed using this signal sequence, as the protein will be fragmented. We have identified a new signal sequence of 18 amino acids from a P. pastoris protein that can secrete proteins to the medium efficiently. The PMT1-gene-inactivated P. pastoris strain secretes a ~30 kDa protein into the extracellular medium. We have identified this protein by determining its N-terminal amino acid sequence. The protein secreted has four DDDK concatameric internal repeats. This protein was not secreted in the wild-type P. pastoris under normal culture conditions. We show that the 18-amino-acid signal peptide at the N-terminal of this protein is useful for secretion of heterologous proteins in Pichia.

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Section: Discussionmentioning

confidence: 99%

A New Signal Sequence for Recombinant Protein Secretion in Pichia pastoris

Govindappa¹,

Hanumanthappa²,

Krishna³

et al. 2014

Journal of Microbiology and Biotechnology

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“…Endogenous signal peptidesCALB and EGFP19, 20 and 23 aa(Liang et al 2013a) Pp Pir1 P.p. Pir1pEGFP and Human α1-antitrypsin61 aa(Khasa et al 2011)HBFI and HBFIIHydrophobins of Trichoderma reesei EGFP16 and 15 aa(Kottmeier et al 2011)…”

Section: Basic Systems For Cloning and Expression In P Pastorismentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

782

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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“…First of all, the structure and linkage differences between β-glucans leaked from yeast cell wall and those in Yeastolate may contribute to the observed β-glucan clearance differences by Protein A purification. A previous report shows that the proteins with internal repeats (PIR proteins) are covalently linked to the β 1,3-glucan of the yeast cell wall via alkali-sensitive ester linkage [25]. Other yeast cell wall constituents such as chitin, manno proteins also play important roles in cell wall integrity and have interactions with β-glucans [26].…”

Section: Discussionmentioning

confidence: 99%

Demonstrating β-glucan Clearance in CHO- and Yeast-Produced Monoclonal Antibodies during Downstream Purification Processes

Wang¹

2014

J Bioproces Biotech

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Biologics production using yeast or CHO with Yeastolate as cell culture additives often introduces β-glucan, which could potentially pose immunogenicity risk, if not adequately removed. Although a previous study has shown the effective clearance of Yeastolate-derived β-glucan by Protein A chromatography, the clearance pattern of yeast cell derived β-glucan remains unknown. In this study, we characterized the β-glucan clearance patterns during downstream processing of three monoclonal antibody (mAb) products, one mAb fragment from Pichia pastoris (mAb A) and two full mAbs from CHO expression system (mAb B and mAb C), by Glucatell assay. We demonstrated effective β-glucan clearance in both small (100 L) and large scale (5000 L) batches of mAb A as well as in one batch of mAb B. Protein A purification step removed an average of 97.74% (1.7 log 10 reduction) of β-glucan detected in the two batches of mAb A microfiltration permeates (MFP) and 99.99% (3.9 log 10 reduction) of β-glucan detected in mAb B clarified culture fluid harvest (HCCF). Residual β-glucans post Protein A purification in the two batches of mAb A were further removed by the two polishing chromatography steps (94.76% reduction on average). Residual β-glucan measured in the mAb A and mAb B drug substance ranges from 7.8 to 19 pg/mg, which is unlikely to alter physiological concentrations significantly in healthy adults when administered with typical intravenous doses. However, in mAb C, after almost complete removal (99.99%) by Protein A purification step, β-glucan level increased more than 20 fold in Viral Filtration (VF) product sample, indicating that it can be introduced from materials used in downstream process, such as cellulose-based filters and membranes. Our study results suggest that although β-glucan can be cleared by Protein A and other chromatographic steps such as AEX, monitoring β-glucan clearance during downstream process development remains very important to identify and avoid potential contaminations to the drug substance.

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Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion

Abstract: Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported.

Cited by 35 publications

References 49 publications

A New Signal Sequence for Recombinant Protein Secretion in Pichia pastoris

A New Signal Sequence for Recombinant Protein Secretion in Pichia pastoris

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Demonstrating β-glucan Clearance in CHO- and Yeast-Produced Monoclonal Antibodies during Downstream Purification Processes

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