A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)
Autor: | Kamini Govender, Anil A. Chuturgoon, Naeem Sheik Abdul, Sooraj Baijnath, Hendrik G. Kruger, Thavendran Govender, Johnson Lin, Tricia Naicker, Taskeen Fathima Docrat |
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Rok vydání: | 2019 |
Předmět: |
Expression vector
Chemistry Insulin medicine.medical_treatment lcsh:Biotechnology Biosynthesis of human insulin Diabetes Biophysics lcsh:QR1-502 E. coli Biological activity medicine.disease_cause medicine.disease Applied Microbiology and Biotechnology In vitro lcsh:Microbiology Biochemistry Diabetes mellitus lcsh:TP248.13-248.65 medicine MTT assay Original Article Escherichia coli Protein Renaturation |
Zdroj: | AMB Express AMB Express, Vol 10, Iss 1, Pp 1-9 (2020) |
ISSN: | 2191-0855 |
Popis: | Insulin has captured researchers’ attention worldwide. There is a rapid global rise in the number of diabetic patients, which increases the demand for insulin. Current methods of insulin production are expensive and time-consuming. A PCR-based strategy was employed for the cloning and verification of human insulin. The human insulin protein was then overexpressed in E. coli on a laboratory scale. Thereafter, optimisation of human insulin expression was conducted. The yield of human insulin produced was approximately 520.92 (mg/L), located in the intracellular fraction. Human insulin was detected using the MALDI-TOF-MS and LC–MS methods. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of human insulin was tested in vitro using a MTT assay, which revealed that the crude biosynthesised human insulin displayed a similar degree of efficacy to the standard human insulin. This study eliminated the use of affinity tags since an untagged pET21b expression vector was employed. Tedious protein renaturation, inclusion body recovery steps, and the expensive enzymatic cleavage of the C-peptide of insulin were eliminated, thereby making this method of biosynthesising human insulin a novel and more efficient method. |
Databáze: | OpenAIRE |
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