Expression of human epidermal growth factor using Ssp DnaB mini-intein as fusion partner in Escherichia coli BL21(DE3)

Abstract

The human epidermal growth factor (hEGF) is widely used clinically as a wound healer, as it has a vital role in stimulating cell proliferation, differentiation, and migration. Consequently, the large-scale production of recombinant hEGF in E. coli has been developed to meet the high demand for hEGF clinically. However, intracellular proteins in E. coli, especially small proteins like hEGF, are degraded by proteases. To overcome this issue, hEGF was fused with CBD-Ssp DnaB to construct a fusion protein CBD-Ssp DnaB-hEGF. This study was conducted to obtain refolded hEGF from the inclusion bodies of CBD-Ssp DnaB-hEGF. The experiment was carried out using E. coli BL21(DE3) containing plasmid pD861-CBD-Ssp DnaB-hEGF. The CBD-Ssp DnaB-hEGF gene was constructed by fusing CBD-Ssp DnaB and hEGF gene and then was optimized. The method was started with E. coli transformation, CBD-Ssp DnaB-hEGF expression, inclusion bodies solubilization, refolding, and simultaneous cleavage to release hEGF. The CBD-Ssp DnaB-hEGF was expressed as inclusion bodies, which can then be purified by washing with Triton X-100 and 1 M urea. The inclusion bodies were solubilized in 8 M urea, the solubilized CBD-Ssp DnaB-hEGF was reformed by dialysis, and then hEGF was spliced by shifting the pH from 8.5 to 6.0 to yield a concentration of 0.163 mg/ml. Therefore, we concluded that hEGF was obtained from the solubilized CBD-Ssp DnaB-hEGF from inclusion bodies produced by E. coli BL21(DE3).

Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity

“…The widely used oxido-shuffling agent, glutathione in reduced and oxidised form was chosen to help the formation of correct disulphide bonds ( Gieseler et al., 2018 ; Thomson et al., 2012 ), as it speeds up the bond formation of hEGF ( Chang et al., 2001 ). Ssp DnaB mini-intein cleavage was performed by lowering the pH during dialysis ( Yosua et al., 2021 ) to induce cyclisation of Asp154 at the C-terminal of Ssp DnaB mini-intein through a relay system involving some amino acid residues ( Ding et al., 2003 ). It causes mini-intein cleavage and release of hEGF.…”

“…The recombinant plasmid was constructed by our team and synthesized by ATUM (Newmark, CA, USA). The CBD- Ssp DnaB-hEGF was successfully expressed by Yosua et al. (2021) as a 31.4 kDa protein.…”

Overexpression of soluble recombinant Thermus thermophilus (Tth) DNA polymerase in Escherichia coli BL21(DE3) using an MBP fusion tag as a solubility enhancer

“…To determine the optimal expression of the soluble fraction, semiquantitative densitometry analysis was carried out using ImageJ (Yosua et al, 2021), indicating that the highest yield of His-Tth DNA polymerase was achieved when the induction was carried out in the mid-log phase (Fig. 4a), whereas induction in the mid-log phase provided a high yield due to the rapid growth of E. coli leading to a higher rate of protein expression (Yosua et al, 2021). Meanwhile, the optimal induction of MBP-Tth DNA polymerase was in the late log phase (Fig.…”