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

Iman Permana Maksum Diandra Firdiani Utami Eva Annisa Nurhakim Yosua Sriwidodo Muhammad Yusuf Muhammad Fadhlillah Ryan Adibagus Haryanto   

Open Access   

Published:  Jun 13, 2022

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.


Keyword:     Escherichia coli BL21(DE3) pD861-His pD861-MBP RTPCR Tth DNA polymerase.


Citation:

Maksum IP, Utami DF, Nurhakim EA, Yosua Y, Yusuf M, Fadhlillah M, Haryanto RA. Overexpression of soluble recombinant Thermus thermophilus (Tth) DNA polymerase in Escherichia coli BL21(DE3) using an MBP fusion tag as a solubility enhancer. J Appl Pharm Sci, 2022. Online First.

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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