Detection of soluble co-factor dependent protein expression in vivo : Application to the 4′-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis

Abstract

The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.

Conformational flexibility of coenzyme A and its impact on the post‐translational modification of acyl carrier proteins by 4′‐phosphopantetheinyl transferases

“…Activity assays were performed as previously described [23]. Briefly, PptAb at 20 n m was incubated at 30 °C with 10 μ m of ACP domain of PpsC, 50 µ m CoA, and 10 m m DTT.…”

“…It was produced in Escherichia coli BL21(DE3) cells missing the endogenous PPTase EntD [11] and purified using a one-step protocol (see Materials and methods). We evaluated the effect of pH on the transfer of the Ppant moiety of CoA on the ACP fragment in the presence of either Mg 2+ or Mn 2+ , as previously described [23]. At pH 7, a transfer of the Ppant moiety is detected on a 10% urea/PAGE in the presence of either cation.…”

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

“…This system was of key importance in confirming the increased solubility of the full-length mycobacterial enzyme PptT in E. coli compared to the N-terminal and C-terminal truncated versions. The addition of Co-enzyme A and Mg 2+ ions to the cell lysate was essential to improve the stability of the enzyme in vitro [43]. The bipartite split-GFP can also be used in support to directed evolution protocols to normalize fluorescence values obtained from the screening of variants with improved enzymatic activities [44].…”

Structure, Biochemistry, and Inhibition of Essential 4′-Phosphopantetheinyl Transferases from Two Species of Mycobacteria

“…25 Therefore, we began studying PptT as an N-terminal maltose binding protein (MBP) fusion. In vitro removal of the MBP domain via a thrombin protease cleavage site led to significant precipitation of PptT.…”