Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

Hu, Hong; Gao, Jie; He, Jun; Yu, Bing; Zheng, Ping; Huang, Zhiqing; Mao, Xiangbing; Yu, Jie; Han, Guang; Chen, Shuai

doi:10.1371/journal.pone.0058393

Cited by 67 publications

(41 citation statements)

References 36 publications

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“…Taken together, these observations strongly suggested that KERQ7 was a monomeric protein comparable to those previously reported for other keratinases from Bacillus strains [15,25,27,[29][30][31].…”

Section: Purification and Molecular Weight Determination Of Kerq7 Enzymesupporting

confidence: 88%

“…Lane 3, dialyzed sample obtained after ammonium sulfate fractionation 30-60% (30 g). Lane 4, purified keratinase KERQ7 (30 g) obtained after S Sepharose cation-exchange chromatography (fractions[28][29][30][31][32][33][34][35], and (C) Zymogram activity staining of the purified keratinase using keratin azure as a substrate.Pyrase ® 250 MP, and KERQ7 retained 70, 73, 80, and 90% of their activity at pH 5 after 240 h incubation at 30 • C, respectively. At pH 7, and using keratin azure as substrate, KERQ7, Koropon ® SC 5K, Basozym ® CS 10, and Pyrase ® 250 MP, were optimally active at 25, 30, 30, and 35 • C in the absence of CaCl 2 and at 30, 35, 35, and 40 • C, respectively in the presence of 1 mM Ca 2+…”

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confidence: 99%

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A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process

Jaouadi

Rekik

Elhoul

et al. 2015

International Journal of Biological Macromolecules

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Section: Purification and Molecular Weight Determination Of Kerq7 Enzymesupporting

confidence: 88%

mentioning

confidence: 99%

A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process

Jaouadi

Rekik

Elhoul

et al. 2015

International Journal of Biological Macromolecules

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“…Gene sequence optimization has been a widely effective measure to increase heterologous protein expression levels in P. pastoris (32)(33)(34)(35)(36), but the mechanism behind this strategy has seldom been studied. The results presented herein are consistent with prior insights that premature transcription termination may result from short segments of severe nucleotide bias within some native genes (37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Zhao

Blazanovic

Choi

et al. 2014

Appl Environ Microbiol

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e Lysostaphin represents a promising therapeutic agent for the treatment of staphylococcal infections, in particular those of methicillin-resistant Staphylococcus aureus (MRSA). However, conventional expression systems for the enzyme suffer from various limitations, and there remains a need for an efficient and cost-effective production process to facilitate clinical translation and the development of nonmedical applications. While Pichia pastoris is widely used for high-level production of recombinant proteins, there are two major barriers to the production of lysostaphin in this industrially relevant host: lack of expression from the wild-type lysostaphin gene and aberrant glycosylation of the wild-type protein sequence. The first barrier can be overcome with a synthetic gene incorporating improved codon usage and balanced A؉T/G؉C content, and the second barrier can be overcome by disrupting an N-linked glycosylation sequon using a broadened choice of mutations that yield aglyscosylated and fully active lysostaphin. The optimized lysostaphin variants could be produced at approximately 500 mg/liter in a small-scale bioreactor, and 50% of that material could be recovered at high purity with a simple 2-step purification. It is anticipated that this novel highlevel expression system will bring down one of the major barriers to future development of biomedical, veterinary, and research applications of lysostaphin and its engineered variants. L ysostaphin (LST) is a glycyl-glycine zinc-dependent endopeptidase natively encoded on the pACK1 plasmid of Staphylococcus simulans (1), an environmental competitor of Staphylococcus aureus. LST is synthesized as a preproenzyme of 493 amino acids, and its pre-(36 amino acids) and pro-(211 amino acids) sequences are removed during and after secretion, respectively. Mature LST is a monomer composed of an N-terminal catalytic domain (132 amino acids), a C-terminal cell wall binding domain (102 amino acids), and a short connecting linker (13 amino acids) between the two (2). The LST enzyme selectively and efficiently degrades pentaglycine cross-links in the peptidoglycan component of S. aureus cell walls, ultimately resulting in bacterial lysis and death. Lysostaphin was discovered in the 1960s (3) and has since undergone various degrees of preclinical development and even small-scale clinical testing by different groups and organizations (4-8). Early interest in LST as a therapeutic agent waned as a result of ready access to conventional drugs, such as methicillin, but enthusiasm for LST in biomedical applications has been revived due to wide-spread antibiotic resistance and shallow antimicrobial development pipelines (9).One barrier to LST clinical applications is the high doses required to eradicate some infections. LST appeared to show good efficacy in an unresponsive leukemia patient suffering from multidrug-resistant staphylococcal pneumonia, multiple abscesses, and cellulitis (7), but this effect required a 500-mg systemic bolus of enzyme. In another study, nasal carriers o...

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“…The yeast was transformed with 1 μg of Sac I-linearized pPICZα A-RMlip by the spheroplast method of Cregg et al (1985) Cultivation. Standard cultivation method was carried out according to method described in Hu et al (2013) and Wang et al (2016). A single positive colony o was cultivated in 100 mL of BMGY medium at 30 C overnite under constant agitation at 250 rpm, until an OD = 2-5 was reached.…”

Section: Methodsmentioning

confidence: 99%

Cloning of Synthetic Lipase Gene from Rhizomucor miehei with Original Signal Peptide in Pichia pastoris

Cahyani¹,

Helianti²,

Nurhayati³

et al. 2017

Microbiol Indones

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Lipases (EC 3.1.1.3) are classified as hydrolases that hydrolyze lipids. These enzymes have potential application in biotechnology and industrial process. In previous study we have cloned the synthetic Rhizomucor miehei lipase gene using the vector pUC57 in Escherichia coli DH5α, but only found the very low enzymes activity. This study aimed to clone synthetic Rhizomucor miehei lipase gene into Pichia pastoris expression plasmid for lipase expression with the original signal peptide. A DNA fragment with the original signal peptide had been obtained by PCR, cut by Xho I and Xba I and then ligated into pPICZα A linearized with the same enzymes. The mixture of ligation reaction, then was transformed into Escherichia coli DH5α. Zeocin-resistant transformants were selected and contained plasmid was analyzed by restriction enzymes analyses and DNA sequencing. As the result, a synthetic Rhizomucor miehei lipase gene (RMlip) with the size of 1132 bp was successfully cloned to pPICZα A vector plasmid. The recombinant plasmid with the correct DNA sequence was transformed into Pichia pastoris X33. Cultivation of recombinant P. pastoris was carried out with the addition of 1.5% methanol every day with appropriate aeration. The recombinant lipase produced by Pichia pastoris X33 o containing RMlip in its chromosomal DNA had optimal temperature and pH, 30 C and 9.0, respectively.

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Codon Optimization Significantly Improves the Expression Level of a Keratinase Gene in Pichia pastoris

Cited by 67 publications

References 36 publications

A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process

A novel keratinase from Bacillus tequilensis strain Q7 with promising potential for the leather bating process

Gene and Protein Sequence Optimization for High-Level Production of Fully Active and Aglycosylated Lysostaphin in Pichia pastoris

Cloning of Synthetic Lipase Gene from Rhizomucor miehei with Original Signal Peptide in Pichia pastoris

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