Development of clone with novel TrpE fusion tag in E. coli for overexpression of trypsin in a bench-scale bioreactor.
Prep Biochem Biotechnol
; 51(2): 144-152, 2021.
Article
em En
| MEDLINE
| ID: mdl-32749186
Trypsin is a key enzyme under the serine proteases that is found in the pancreas which plays a key role in protein digestion. It cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine. This enzyme has received greater attention mainly due to its increased use in the removal of fusion tags during protein purification and its role in the processing of biosimilars like insulin. The present study was carried out to develop a clone with Novel TrpLE1413(TrpE) Fusion Tag for enhanced expression of trypsin which helps in cost reduction of biosimilar processing. In our experiment we have used a synthetic bovine trypsin gene containing a novel fusion tag TrpE at its N terminus, which was cloned into the pET41b (+) vector and expressed in E. coli BL21 (DE3) in a lab-scale bioreactor. Using the optimized fermentation process with TrpE Fusion Tag, 27.8 g/L inclusion bodies were produced at the end of fermentation, of which 209 mg/L of active trypsin was obtained after purification. In contrast, previous reports have claimed to produce a maximum of 60 mg/L of the enzyme without the fusion tag. Thus based on our findings, the small size (less than 2 kDa) of TrpE tag and its hydrophobicity may reduce the loss incurred during the purification process. Hence, it could be discerned that the use of the TrpE fusion tag along with a robust fermentation process led to 3- 4 fold higher yield making it a commercially viable process facilitating an improved recovery of the enzyme.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Tripsina
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Corpos de Inclusão
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Reatores Biológicos
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Escherichia coli
Limite:
Animals
Idioma:
En
Revista:
Prep Biochem Biotechnol
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Índia
País de publicação:
Reino Unido