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

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, thereby making this method of biosynthesising human insulin a novel and more efficient method.

Keywords: Biosynthesis of human insulin; Diabetes; E. coli.

Fig. 1

Images displaying the PCR amplicons of proinsulin DNA, purified vector DNA, and the integrated pET21b-hPin vector with the inserted proinsulin gene. Lanes 1 and 12 contain one kb molecular weight marker. Lanes 2 to 7 contains the PCR amplicons of proinsulin flanked with BamHI and XhoI restricted ends; Lane 8 contains the purified pET21b miniprep product; Lanes 9 to 11 contain the integrated pET21b-hPin vector miniprep products

Fig. 2

A MALDI-TOF spectrum illustrating the supernatant sample of crude biosynthesised human insulin, which was induced at 1 mM IPTG

Fig. 3

A peptide spectrum illustrating the protein sequence of crude biosynthesised human insulin, which was a 100% match to the human insulin sequence derived from the Scaffold 1.4.4 software

Fig. 4

A graph illustrating the cell viability of HepG2 cells, under hyperglycaemic conditions for standard human insulin and crude biosynthesised human insulin at low (50 ng/mL), medium (100 ng/mL) and high (150 ng/mL) MTT concentrations (p-value < 0.0001)