Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris

Karaoglan, Mert; Erden-Karaoğlan, Fidan

doi:10.1016/j.enzmictec.2020.109589

Cited by 24 publications

(11 citation statements)

References 37 publications

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“…The codon optimization of foreign genes is a widely used method to improve the protein expression levels in host organism. The common strategy for codon optimization is to replace rare codons with preferred codons to match the CUB of the host organism, which is also widely used in Pichia pastoris [ 14 , 30 , 31 ]. Can the optimized sequences produced from codon-pair context perform better in protein expression than that produced from codon usage in Pichia pastoris ?…”

Section: Resultsmentioning

confidence: 99%

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

Huang

Lin

et al. 2021

Microb Cell Fact

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Background Codon optimization is a common method to improve protein expression levels in Pichia pastoris and the current strategy is to replace rare codons with preferred codons to match the codon usage bias. However, codon-pair contexts have a profound effect on translation efficiency by influencing both translational elongation rates and accuracy. Until now, it remains untested whether optimized genes based on codon pair bias results in higher protein expression levels compared to codon usage bias. Results In this study, an algorithm based on dynamic programming was introduced to develop codon pair optimization (CPO) which is a software tool to provide simple and efficient codon pair optimization for synthetic gene design in Pichia pastoris. Two reporters (MT1-MMP E2C6 and ADAM17 A9B8 scFvs) were employed to test the effects of codon pair bias and CPO optimization on their protein expression levels. Four variants of MT1-MMP E2C6 and ADAM17 A9B8 for each were generated, one variant with the best codon-pair context, one with the worst codon-pair context, one with unbiased codon-pair context, and another optimized based on codon usage. The expression levels of variants with the worst codon-pair context were almost undetectable by Western blot and the variants with the best codon-pair context were expressed well. The expression levels on MT1-MMP E2C6 and ADAM17 A9B8 were more than five times and seven times higher in the optimized sequences based on codon-pair context compared to that based on codon usage, respectively. The results indicated that the codon-pair context-based codon optimization is more effective in enhancing expression of protein in Pichia pastoris. Conclusions Codon-pair context plays an important role on the protein expression in Pichia pastoris. The codon pair optimization (CPO) software developed in this study efficiently improved the protein expression levels of exogenous genes in Pichia pastoris, suggesting gene design based on codon pair bias is an alternative strategy for high expression of recombinant proteins in Pichia pastoris.

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Section: Resultsmentioning

confidence: 99%

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

Huang

Lin

et al. 2021

Microb Cell Fact

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“…21 This finding is in agreement with previous reports on the acidic nature of their extracted polygalacturonase. [21][22][23] Figure 4 shows the optimum temperature of polygalacturonase activity of Chrysophyllum albidum fruit extract was 40°C.…”

Section: Resultsmentioning

confidence: 99%

Partial Purification and Kinetic Properties of Polygalacturonase from Chrysophyllum albidum G. Don Fruit

2021

TJNPR

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Over the years, pectinases have been produced primarily through fermentation with fungal cultures of Aspergillus, Penicillium, Bacillus and Trichoderma species and other sources such as bacteria and yeasts. [12][13][14][15][16] Plant pectinases have high activity and stability in a wide range of temperature and pH. 17 These properties have intensified interest in seeking plant sources of pectinase with good properties for industrial use, which could lead to a more efficient and cheaper yield of fruit juice. Therefore, the present study seeks to characterize and determine the kinetic properties of polygalacturonase from the fruit extract of Chrysophyllum albidum. Materials and Methods Chemicals and ReagentsAll chemicals and reagents of analytical grade were obtained from Sigma-Aldrich Chemie, Klincent and Qualikems Laboratory.

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“…The prepared samples were run in a 5% polyacrylamide loading gel and 10% polyacrylamide separation gel in 1xTGS buffer (0.025 M Tris base, 0.192 M Glycine, 0.1% SDS, pH 8.3) at 100 V for 90 minutes to separate proteins. The gel was then stained with Coomassie Brilliant Blue dye and the proteins were visualized after the dye was removed (Karaoğlan and Erden-Karaoğlan, 2020).…”

Section: Sds-page Analysismentioning

confidence: 99%

“…The resin obtained was washed 3 times with 1xPBS containing 25 mM imidazole, and then the protein (glucanase) attached to the resin was treated 3 times with 1xPBS elution buffer containing different concentrations of imidazole (100, 200 and 400 mM). Samples obtained from each step of the purification procedure were analyzed for determination of the quality of purification by SDS-PAGE analysis (Karaoğlan and Erden-Karaoğlan, 2020). The total amount of protein in the elution samples was measured using the Coomassie Bradford Plus Protein Assay Kit following the kit protocol.…”

Section: Purification Of the Recombinant Glucanase Enzyme By Affinity Chromatography (Ni-nta)mentioning

confidence: 99%

“…The glucanase gene from Bacillus licheniformis was expressed in P. pastoris with codon optimization, and an approximately 10-fold (mg/L) increase was reported compared to codon non-optimized expression (Teng et al, 2007). It is clear from previous studies that codon optimization of the glucanase gene (Luang et al, 2010;Pham et al, 2011) and also others (Wang et al, 2015;Qiao et al, 2010;Karaoğlan and Erden-Karaoğlan, 2020) has a significant effect on the expression level of P. pastoris. R. miehei glucanase gene was first isolated by cDNA synthesis and its gene sequence was revealed (Tang et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

Karaoglan

2021

Erzincan Üniversitesi Fen Bilimleri Enstitüsü Dergisi

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In this study, Rhizomucor miehei -1,3-1,4-glucanase gene was expressed under the regulation of AOX1 promoter in the Pichia pastoris expression system, and the clone providing the highest production level was determined. The codon-optimized gene was ligated into expression vector pPICZA and transferred into competent P. pastoris KM71H cells. Thirty transformants were selected from plates containing different concentrations of zeocin and cultured in test tubes for protein production. Glucanase enzyme activities in supernatant samples were measured and ten clones showing the highest glucanase activity were determined. The proteins in supernatant samples were also analyzed by SDS-PAGE and the glucanase enzyme was observed in 2 bands, approximately 34 and 38 kDa. Protein production was performed for 72 hours in 200 mL induction medium (BMMY) with the clone providing the highest glucanase enzyme production and the recombinant glucanase enzyme was purified by Ni-NTA affinity chromatography. After purification, it was determined that the analyzed clone reached 79.6 mg/L glucanase production level. SDS-PAGE analysis of samples from each step of the purification procedure showed that the two protein bands also observed in the supernatant samples represent glucanase enzyme.

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Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris

Cited by 24 publications

References 37 publications

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

Codon pair optimization (CPO): a software tool for synthetic gene design based on codon pair bias to improve the expression of recombinant proteins in Pichia pastoris

Partial Purification and Kinetic Properties of Polygalacturonase from Chrysophyllum albidum G. Don Fruit

Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

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