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.

Keywords: 4′-Phosphopantetheinyl transferase; 4′-phosphopantetheine; ACP; AcpS; AnTet; BSA; Co-factor; CoA; Coenzyme A; DHFR; DSF; DTT; Domain trapping; GFP; IMAC; IPTG; Kan; LB; Luria–Bertani; MBP; P-pant; PCR; PDIM; PKS; Polyketide; SDS–PAGE; SEC; SUMO; Sfp; Spec; Split-GFP; Tuberculosis; acyl carrier protein; acyl carrier protein synthase; anhydrotetracycline; bovine serum albumin; differential scanning fluorimetry; dihydrofolate reductase; dithiothreitol; green fluorescent protein; immobilized metal ion affinity chromatography; isopropyl β-d-1-thiogalactopyranoside; kanamycin; maltose binding protein; phtiocerol dimycocerosate; polyketide synthase; polymerase chain reaction; size-exclusion chromatography; small ubiquitin-related modifier; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; spectinomycin; surfactin synthetase-activating enzyme.