Recent advances on the GAP promoter derived expression system of Pichia pastoris

Zhang, Ailian; Luo, Jinxian; Zhang, Tianyuan; Pan, Ying-Wen; Tan, Yanhua; Fu, C. Y.; Tu, Fa-Zhi

doi:10.1007/s11033-008-9359-4

Cited by 142 publications

(111 citation statements)

References 51 publications

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“…In all three constructs the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was used to drive expression (13,14). To ease the purification and to minimize internal proteolytic activity the constructs were designed for secretion.…”

Section: Resultsmentioning

confidence: 99%

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Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

et al. 2015

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A method to express, purify and modify the PeptidylLys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S 1 0 binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S 1 0 , it was proposed to extend the S 1 0 binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P 1 0 . Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S 2 0 S 4 0 compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.Keywords: enzyme kinetics/enzyme mechanism/ metalloprotease/molecular modelling/site-directed mutagenesis.Abbreviations: Am-LysN, Armillaria mellea peptidyl-Lys metallopeptidase; DsbA, disulfideoxidoreductase; FRET, Fo¨rster resonance energy transfer; GAP, glyceraldehyde-3-phosphatedehydrogenase; IPTG, isopropylb-D-1-thiogalactopyranoside; MOE; Molecular Operation Environment (CCG); MOPS, 3-(N-morpholine)propanesulfonic acid; MS, mass spectrometry; PCR, polymerase chain reaction; RMSD, root-mean-square deviation; SDSPAGE, sodiumdodecylsulfatepolyacrylamide gel electrophoresis.In this article, we use Schechter and Berger nomenclature (1).Peptidyl-Lys metallopeptidase (LysN) from Armillaria mellea is an aspzincin metalloprotease, which cleaves in front of lysines (2). Hence, it is unusual by having its substrate specificity at the C-terminal side of the scissile bond (/K) (3). The protease is predominantly used as a tool in proteomic studies, where it serves as an alternative or complement to trypsin. The advantage of LysN is that it generates fragments which have a positive charge at the N-terminus, yielding mass spectrometry (MS) fragmentation with clear b-ion ladders (4,5). This feature can be used to facilitate de novo peptide mapping. Recently we profiled the substrate specificity of AmLysN and found it to be strictly lysine specific at S 1 0 , and having decreasing specificity when moving away from the scissile bond (2, 6).We also reported a homology model of Am-LysN based on the crystal structure of Gf-LysN (7). The two structures had a RMSD of 0.001 on backbone residues and 0.239 on the side chain residues (2). In this model Am-LysN has a narrow, and relatively short (57 amino acid) binding cleft. Central to the hydrolysis of peptides, is the zinc ion and the zinc binding motif 'HExxH + D'. Fushimi et al. showed that the aspartic acid D132 is necessary to coordinate zinc binding of the homologous protein ...

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Section: Resultsmentioning

confidence: 99%

“…The lysn was under control of the T7 promoter and is flanked by the T7 terminator (12). pAAR4, pAAR5, pAAR6 were constructed by cloning the genes into a vector under control of the P. pastoris GAP promoter and flanked by a GAP terminator (13,14).…”

Section: Construction Of Expression Plasmidsmentioning

confidence: 99%

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

et al. 2015

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“…Moreover, GAPDH mRNA accounts for 2-5% of the poly(A) RNA in yeasts, suggesting that the GAPDH gene is driven by a highly active promoter [5]. General vectors have also been designed that rely on homologous GAPDH promoter regions to drive the expressions of heterologous genes in yeasts [28], filamentous fungi [18,20] and mushroom [12]. The GAPDH gene has also been cloned from some algae such as Chlamydomonas sp.…”

Section: Introductionmentioning

confidence: 99%

A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Promoter for Expressing Transgenes in the Halotolerant Alga Dunaliella salina

Jia

Allen

et al. 2012

Curr Microbiol

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A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.

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“…Full transcription from P GAP is achieved when grown in glucose as a carbon source. 13,14 In addition to P GAP other moderately strong constitutive systems may be used such as P TEF1 15 and P PGK1 .…”

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confidence: 99%

Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

et al. 2017

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Komagataella phaffii (formerly Pichia pastoris) is a well-known fungal system for heterologous protein production in the context of modern biotechnology. To obtain higher protein titers in this system many researchers have sought to optimize gene expression by increasing the levels of transcription of the heterologous gene. This has been typically achieved by manipulating promoter sequences or by generating clones bearing multiple copies of the desired gene. The aim of this work is to describe how these different molecular strategies have been applied in K. phaffii presenting their advantages and drawbacks.

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Recent advances on the GAP promoter derived expression system of Pichia pastoris

Cited by 142 publications

References 51 publications

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Promoter for Expressing Transgenes in the Halotolerant Alga Dunaliella salina

Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

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