Expression of Mycobacterium tuberculosis Protein Tyrosine Phosphatase B in Escherichia coli and Its Recovery from Inclusion Body

Abstract

The present study aims at expressing and partially purifying PtpB in active form. To achieve this, Mtb PtpB gene has been cloned into pET30a vector and overexpressed in Escherichia coli BL 21(DE3) under IPTG induction in the form of an inclusion body. Following resolubilization by urea and dialysis, the resulted PtpB has been shown to be active against para-Nitrophenyl phosphate. It is concluded that the resulted PtpB has had been recovered from inclusion body to give the active form of the enzyme, and thus the success in overexpressing PtpB provides the required material to investigate the biochemical properties of the pathogen virulence factor further.

cis-2 and trans-2-eicosenoic Fatty Acids Inhibit Mycobacterium tuberculosis Virulence Factor Protein Tyrosine Phosphatase B

“…As shown in Figure 1B, re-solubilization with 6 M GuHCl followed by dialysis effectively removes the excessive salt. The application of GuHCl gave a better result compared to previous work with 8 M urea (24). The observation might come from the relatively efficient solubilization of the IB by GuHCl compared to urea (25).…”

“…Recovery of PtpB from inactive IB was made resolubilization and dialysis against refolding buffer, as described elsewhere (24). Briefly, IB was washed three times with wash buffer (50 mM Tris pH 8; 0.1 mM NaCl; 0.1 mM EDTA, 5% glycerol; 0.1 mM DTT, and 5% Triton X-100).…”

Effect of Ethanol and IPTG on the Recombinant Jembrana Trans-Activator of Transcriptation Protein Expression

“…By adding IPTG, the expression of T7 RNA polymerase fused in the chromosome of E. coli is increased. The result T7 RNA polymerase recognizes the T7 promoter at the upstream position of the target gene (Savalas et al, 2017).…”