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Papain expression in the Escherichia coli cytoplasm by T7-promoter engineering and co-expression with human protein disulfide isomerase (PDI) and thiol peroxidase (GPx7) genes
Sunday, 2025/02/23 | 06:35:53
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Md Anarul Hoque, Richard A Gross, Mattheos A G Koffas Appl Environ Microbiol.; 2025 Jan 31; 91(1):e0211924. doi: 10.1128/aem.02119-24. AbstractDifficulties exist in obtaining full-length, correctly folded, and soluble papain or papain-like proteases that necessitate the exploration of alternative strategies. This study describes the development of an Escherichia coli strain capable of producing soluble papain without the need for complex and time-consuming in vitro refolding steps. To enhance the production of soluble papain, engineered T7 promoters and a recombinant papain translationally fused with varying tags were constructed. The tags investigated include the maltose-binding protein, small ubiquitin modifier protein, and glutathione transferase. An E. coli SHuffle strain was engineered to accumulate hydrogen peroxide (H2O2) by disruption of the redox pathway. This was accomplished by co-expression of the fusion constructs with two human endoplasmic reticulum-resident proteins, thiol peroxidase glutathione peroxidase-7 (GPx7), and protein disulfide isomerase (PDI). The oxidizing capacity of H2O2 was used to improve disulfide bond formation in papain. The GPx7-PDI fusion dyad played a significant role in consuming harmful H2O2 generated by the SHuffle cells. This consumption of H2O2 helped provide the necessary oxidizing conditions for the efficient production of soluble papain. In shake-flask experiments, the recombinant strain produced ~110 mg/L of papain. Moreover, in batch fermentation, the volumetric yield reached ~349 mg/L. This work provides insights into recombinant papain microbial production that can lead to an industrial viable production strain.
Importance: Papain, a cysteine-like protease, has extensive applications across various industries including food, chemical, pharmaceutical, drug, and polymer. However, the traditional isolation of papain from Carica papaya plants results in a complex mixture of proteases. Such protease mixtures result in an inability to understand which component enzyme contributed to substrate conversions. Concentrations of constituent enzymes likely differ based on the ripeness of the papaya fruit. Also, constituent enzymes from papaya differ in optimal activity as a function of temperature and pH. Thus, by using papain-like enzymes from papaya fruit, valuable information on component enzyme activity and specificity is lost. Numerous methods have been reported to purify papain and papain-like enzymes from the crude mixture. Often, methods involve at least three steps including column chromatography to separate five cysteine proteases. Such procedures represent tedious processes to manufacture the pure enzymes in Carica papaya extracts. The numerous uses of papain for industrial processes, as well as the probability that certain components of papain crude mixtures will be preferred for specific applications, necessitate alternative methods such as recombinant expression from microbial production systems to meet the high world demand for papain.
See https://pubmed.ncbi.nlm.nih.gov/39589110/
Figure 1: Disulfide bond formation in Escherichia coli expression systems. (A) The E. coli cytoplasm is generally reduced due to high levels of reduced glutathione (GSH) relative to oxidized glutathione (GSSG), maintained by glutathione reductase (gor) and thioredoxin reductase (TrxB). In the periplasm, DsbA facilitates disulfide bond formation. DsbB regenerates DsbA by oxidizing it. Misfolded proteins with incorrect disulfide bonds are processed by DsbC, which requires its redox state to be maintained by DsbD. (B) SHuffle E. coli has been engineered to create a more oxidizing environment in the cytoplasm. This is achieved by knocking out Gor (reducing the GSH ratio) and TrxB (preventing reduction of Trx1). The reduced ability to maintain a high level of reduced glutathione and the lack of TrxB shift the redox state toward oxidation, allowing disulfide bond formation to occur within the cytoplasm. SHuffle E. coli is engineered to cytoplasmically express DsbC, which can isomerize incorrectly disulfide-bonded proteins in the cytoplasm. (C) CyDisCo retaining protein disulfide isomerase (PDI)-sulfhydryl oxidase (Erv1P); co-expression in SHuffle E. coli further strengthens the oxidation pathway in the cytoplasm. CyDisCo co-expression of PDI readily oxidizes newly translated proteins in the cytoplasm. The redox state of PDI is reset by the sulfhydryl oxidase Erv1p, which generates de novo disulfide bonds by donating electrons on to O2 and form H2O2. (D) pACYC-duet retaining PDI-glutathione peroxidase-7 (Gpx7) co-expression in SHuffle of the PDI-GPx7 system mediates disulfide transfer from accumulated H2O2 to recombinant papain via PDI-Gpx7 fusions. |
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