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. 2014 Oct 30;10(10):e1004446. doi: 10.1371/journal.ppat.1004446

Figure 1. ESAT-6 protein interacts with β2M.

Figure 1

(A) Interaction between ESAT-6 and β2M in a yeast two-hybrid system was studied by mating yeast strain AH1109 transformed with bait plasmid pGBKT7-ESAT-6 with yeast strain Y187 transformed with a human leukocyte cDNA prey library on a synthetic dropout plate (–Ade/–His/–Leu/–Trp). Prey cDNA was amplified by PCR using primers encompassing the cDNA insert in pACT2 and sequenced and identified to be β2M. The AH109 yeast strain transformed with pGBKT7-ESAT-6 and pACT2-β2M shows Ade and His interaction reporter activation on QDO plates. AH109 transformed with pGBKT7 and pACT2-β2M was used as a negative control while AH109 transformed with pGBKT7-p53 and pGADT7-T was used as a positive control for the yeast two-hybrid screening. (B) Untagged β2M was cloned along with His-tagged ESAT-6 in the pETDuet vector system and was transformed into E. coli BL21 cells. The transformed cultures were induced with IPTG and the over-expressed proteins were purified using TALON resin. Purified proteins were separated on a 16% Tris-Tricine SDS-PAGE and visualized by silver staining. Lane 1 is a molecular weight marker. (C) β2M with no N-terminal signal sequence (ESAT-6:β2M NSS) and the full length β2M with the N-terminal signal sequence (ESAT-6:β2M WSS) were cloned in pETDuet vector along with 6× His-tagged ESAT-6. Clones were over-expressed in E. coli BL21. Protein expression was induced by addition of IPTG and over-expressed proteins were purified using metal affinity TALON resin. Purified proteins were separated on 16% Tris-Tricine SDS-PAGE and transferred onto nitrocellulose membranes and probed with anti-β2M Ab to detect β2M and anti-His Ab to detect ESAT-6. The bands corresponding to β2M (upper panel) and ESAT-6 (lower panel) were visualized by chemiluminescence. (D) GST pre-cleared THP-1 macrophage extract was mixed and incubated with Glutathione-agarose bead bound-GST or GST-ESAT-6 and washed. The bound proteins were eluted and resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted for β2M protein using rabbit anti-human β2M Ab and HRP conjugated anti-rabbit Ab. Blots were visualized by chemiluminescence. Whole cell extracts from THP-1 cells was used as positive control for β2M expression. (E) In another experiment, purified recombinant 6× His-tagged ESAT-6 was incubated with THP-1 macrophage lysate and immunoprecipitated (IP) with anti-His Ab bound to protein A/G agarose beads. Control immunoprecipitation was carried out without the addition of His-tagged ESAT-6 protein (IP control). The eluted protein mixture is resolved on a 16% Tris-Tricine SDS-PAGE gel and immunoblotted (IB) for β2M protein using a rabbit anti-human β2M Ab and HRP conjugated anti-rabbit secondary Ab and the blots were visualized by chemiluminescence. Lane 1 is input control. (F) Direct interaction of β2M with ESAT-6 was monitored using a BIACORE 3000 Biosensor where β2M was immobilized on the sensor chip and recombinant ESAT-6 at different concentrations was injected in the running buffer. The changes in the refraction index at the surface due to interactions between immobilised β2M and fluid phase ESAT-6 were detected and recorded as RU (Resonance Units). Curves generated from the RU trace were evaluated using a curve-fitting algorithm. ESAT-6 was found to bind specifically to β2M and no binding was observed in a control cell which did not have any immobilized β2M. Results are representative of three different experiments.