Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris

Liang, Shuli; Li, Cheng; Ye, Yanrui; Lin, Ying

doi:10.1007/s10529-012-1055-8

Cited by 39 publications

(24 citation statements)

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“…The combination of cis-acting elements with P AOX1 , a deletion of P AOX1 nucleotides −777 and −712 and the addition of −203 and −190 [ 10 ], was performed to create plasmid pAOX1 d1+201 -α-phy-HKA (AOXm). Based on this plasmid, a 10-residue spacer peptide (EEAEAEAEPK) between the α -factor prepro-signal [ 13 ] and the phytase gene was introduced to create plasmid pAOX1 d1+201 -αE10-phy-HKA (αE10); a deletion of the predicted third alpha helix of the α -factor [ 12 ] was performed to create plasmid pAOX1 d1+201 -α∆57-70-phy-HKA (α∆57-70); and replacement of the α -factor by the signal peptide of Dse4p [ 14 ] was undertaken to create plasmid pAOX1 d1+201 -D-phy-HKA (SP-D). Based on the plasmid αE10, plasmids pPICZA-αE10-HKA/(Phy) 2 (2c), pPICZA-αE10-HKA/(Phy) 4 (4c), and pPICZA-αE10-HKA/(Phy) 6 (6c) were created, which contained two, four and six expression cassettes respectively (Additional files 1 and 3 ).…”

Section: Resultsmentioning

confidence: 99%

“…The vectors pPICHKA and pPICZA-HAC1 were gifts from Dr. Han (South China University of Technology) [ 22 ]. The plasmid pTEFZA-EGFP-HIS-G, which contained the GAPDH gene fragment, was from a previous study [ 14 ]. Strains, vectors and primers used in the present study are summarized in Additional file 2 .…”

Section: Methodsmentioning

confidence: 99%

“…The Saccharomyces cerevisiae α -factor prepro-signal, consisting of a 19-amino acid pre-region signal peptide and a pro-region of 60 hydrophilic amino acids [ 11 ], is the most widely and successfully used secretion signal peptide in P. pastoris . Optimizing the structure of the α -factor [ 12 , 13 ] and choosing endogenous signal peptides, such as the signal peptides of Dse4p [ 14 ], may enhance the export of heterologous protein expressed in P. pastoris . Moreover, gene copy number also influences the expression level efficiency [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

et al. 2015

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BackgroundPhytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.MethodsTo improve the phytase expression, we use modification of PAOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1i to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.ResultsThe phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (PAOX1) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of PAOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12 %, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141 % higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1i (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412 % higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.DiscussionThe production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.ConclusionsUsing modification of PAOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1i to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412 % relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0204-2) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

et al. 2015

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“…leucin-rich peptideHuman lysozyme18 and 16 aa(Oka et al 1999)K28 pre-pro-toxinK28 virus toxinGreen fluorescent protein36 aa(Eiden-Plach et al 2004)Scw, Dse and Exg P.p. 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

803

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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“…Therefore, the intrinsic commercial value of heterologous proteins has driven a wide range of studies on optimizing yeast secretion systems as “cell factories” [ 4 ]. Most studies on yeast secretion systems focused on the vector systems [ 5 ], the host strain [ 6 ], or its cultivation conditions [ 7 ], especially promoters [ 8 , 9 ], signal peptides [ 10 , 11 ], codon usage [ 12 , 13 ], gene copy number [ 14 ], proteases [ 15 ], and chaperones [ 16 ]. Although extensive trials have been conducted, in some cases, secretion of the product into the culture supernatant remains low for some proteins [ 1 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy

Wang

Huang

et al. 2015

Microb Cell Fact

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BackgroundAlthough Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.ResultsThe results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.ConclusionsThese results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0315-4) contains supplementary material, which is available to authorized users.

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Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris

Cited by 39 publications

References 14 publications

Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

Combined strategies for improving expression of Citrobacter amalonaticus phytase in Pichia pastoris

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

Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy

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