A novel and more efficient biosynthesis approach for human insulin production in Escherichia coli (E. coli)

AMB Express
Kamini GovenderThavendran Govender

Abstract

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, thereb...Continue Reading

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Citations

Jul 2, 2021·Computational and Structural Biotechnology Journal·Andrew WalakiraMiha Moškon

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Methods Mentioned

BETA
PCR
protein folding

Software Mentioned

Proteome Discoverer
Graph pad
stat
FlexAnalysis
Scaffold
FlexControl

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