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

Abstract

Tth DNA polymerase is a thermostable enzyme derived from Thermus thermophilus and acts as a DNA polymerase and reverse transcriptase. Escherichia coli is used for large-scale enzyme production because of its cost-effectiveness, rapid growth, and increased recombinant protein expression, but inclusion bodies can be formed during intracellular protein expression, so the maltose-binding protein (MBP) tag was used to improve the expression of soluble protein. The Tth DNA polymerase gene was optimized to have a codon adaptive index of 1.00% and 60.64% Guanine and Cytosine (GC) content, then inserted into E. coli BL21(DE3), which harbors pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase. The induction and postinduction incubation time were optimized to express pD861-His-Tth DNA polymerase and pD861-MBP-Tth DNA polymerase in the soluble form. The total protein concentration of His-Tth DNA polymerase is 3.9095 mg/ml while for MBP-Tth DNA polymerase it is 33.541 mg/ml; protein levels after optimization based on densitometry analysis show MBP-Tth DNA polymerase is seven times higher than His-Tth DNA polymerase. This indicates that MBP tag fusion increases the amount of soluble protein produced.

Manipulation Strategy to Increase Expression Level of Soluble Recombinant Protein Penicillin G Acylase (PGA) in Bacterial Host Escherichia coli: A Review Article

“…Secondly, MBP's role is transient, temporarily preventing improper folding of the recombinant protein until spontaneous folding occurs with the assistance of endogenous chaperones (Bhatwa et al, 2021;Paraskevopoulou & Falcone, 2018). Studies by Maksum et al, (2022) and Fang et al, (2018) have reported that overexpressing heterologous recombinant proteins in the E. coli BL21(DE3) host, in conjunction with MBP sequences in the recombinant plasmid construct, significantly enhances solubility and stability of protein expression compared to traditional overexpression methods without MBP insertion.…”

“…Proteins folding incorrectly may interact with each other, rendering the protein biologically inactive. This issue arises due to the reducing conditions within the cytosol of E. coli and the limited availability of chaperone proteins, which fail to cope with the high levels of recombinant protein expression (Maksum et al, 2022). The challenge of ensuring proper protein conformation during expression in the host is critical in achieving substantial bioactive PGA recombinant protein mass.…”