Project Associates/Assistants
Vidita Chaudhary
Nidhi Dogra
Nimisha Suman
Shaloo Gupta
Aparna Dixit

PhD Students
Pankaj Alone
Gunjan Malik
Deepika Mathur
Divya Mathur

Collaborators
Om Singh

The goal of this project is to clone and express genes of biomedical importance and to understand the molecular mechanisms involved in the regulation of gene expression.

To clone the functionally significant or immunodominant regions of gp63, in C-terminal fusion with LTB, a master clone containing (His)6-tagged LTB in pQE32 vector, with its stop codon removed, was used. Synthetic oligonucleotides spanning the catalytic and binding regions of L. donovani gp63 were commercially synthesized with specific restriction overhangs. Annealed oligonucleotides were ligated to the master clone generating the double epitope fusion pQltbCatB (with catalytic and binding region of gp63). The positive clone was screened by restriction digestion and confirmed by DNA sequencing. The sequence of the LTB fusion constructs has been submitted to the GenBank with the Accession No. AF359362.

Upon induction with IPTG, E. coli M15 cells carrying the recombinant plasmid pQLtbCatB showed the production of His-tagged fusion protein LtbCatBIB of around 17 kDa. The protein was localized in the insoluble inclusion bodies in the cell cytoplasm, which was then solubilized in a buffer containing 6M GuHCl for its purification through metal-affinity chromatography using Ni2+-NTA resin. About 98% pure fusion protein could be obtained.

For secretory expression, LTB-fusion was cloned into secretory expression vector generating the clone pMLtbCatB which carries the LTB gene in C-terminal fusion with gp63 epitopes under the control of LTB signal sequence (LTBss) at the 5’ end. Histidine tag was removed from the fusion protein during this cloning. E. coli cells carrying the recombinant LTB-fusion in pMMB vector were induced with 1 mM IPTG. The fusion protein, LtbCatBPP was getting expressed and localized in the periplasmic space in E. coli.

The recombinant construct was then conjugally transferred from E. coli to V. cholerae host, with the help of pRK2013 strain generating the clones VltbCatB. The fusion gene in V. cholerae cells carrying VltbCatB construct were induced with 1 mM IPTG. The protein was produced in large amounts and efficiently processed and secreted into the extracellular milieu. The native fusion protein LtbCatBSEC was purified from induced V. cholerae culture supernatant by ammonium sulphate cut and ion-exchange chromatography using phosphocellulose column. The fusion protein could be purified to near homogeneity.

The purified proteins LtbCatBIB and LtbCatBSEC could be picked up with anti-LTB antibodies on immunoblot. The purified fusion protein from V. cholerae culture supernatant had the ability to pentamerise in non-denaturing condition. The ability of the fusion protein pentamer to bind to GM1 ganglioside receptor was checked on a sandwich ELISA. The ability of the native LTB fusion protein to pentamerise and bind to GM1 ganglioside receptor on ELISA indicated the possibility of the carrier protein LTB to retain its functional activity after C-terminal fusion.

Polyclonal rabbit anti-gp63 antibodies, raised against pure gp63, could pick up the fusion proteins in immunoblots. Kala azar patient serum could also pick up both the fusion proteins in ELISA and blots.

Both the purified fusion proteins LtbCatBIB and LtbCatBSEC were used to immunize NZW rabbits subcutaneously in CFA. High levels of fusion protein specific antibodies were generated. The rabbit antisera could pick up the fusion proteins in immunoblots. The antibodies produced by the rabbits immunized with LtbCatBIB contained significantly high levels of catalytic domain specific antibodies, as seen in ELISA using synthetic peptides.

The antibody titers for anti-LtbCatBIB did go down with time, though significant titers were maintained upto 20 months post booster indicating a long lasting response to the antigen, without the requirement of a booster for more than a year. However, another booster of the fusion protein in IFA, 25 months post-booster, could again generate high titer antibodies against the fusion protein as well as the catalytic peptide, as detected by ELISA.

Concentrated preparation of Leishmania donovani membrane was used as a crude extract of gp63 for immunoblotting with rabbit antisera. A band at 63 kDa could be obtained with antibodies against both the fusion proteins, thus indicating the ability of the anti-fusion protein antibodies to recognize whole gp63 molecule in the parasite membrane preparation. Mouse anti-LtbCatBIB sera showed a predominance of IgG2a subtype in ELISA, indicating a dominantly Th1 influenced response, whereas mouse anti-LtbCatBSEC sera had higher titers of IgG1 isotypes as compared to IgG2a.

Indirect immunostaining was carried out to check the binding of anti-fusion protein antisera to Leishmania cell surface gp63. Both the fusion protein antisera decorated the whole flagellar membrane and cell body of promastigotes. No detectable staining could be observed with preimmune serum or rabbit anti-LTB serum.

To study the effect of fusion protein antisera on the proteolytic activity of gp63, cells were pre-incubated with increasing concentrations of purified anti-fusion protein IgG and then activity was checked at different time points. A drop in the rate of activity could be observed with increasing concentration of IgG used. As much as 60-70% inhibition in surface gp63 protease activity could be seen with IgG treatmtent. This indicates a blockage in the catalytic domain(s) of gp63 on the surface of live Leishmania promastigotes by purified anti-fusion protein IgGs, thus interfering in its proteolytic activity.

Cells, pre-treated with increasing concentrations of IgG, were incubated with 10% NGPS and percentage of viable cells was determined by MTT assay. Upon lysis in 5% NGPS, a decrease in the number of viable cells could be observed with increasing amounts of IgG used. 50-70% drop in the percentage of viable cells could be observed upon incubation with 1 mg of IgG as compared to untreated promastigotes or promastigotes incubated with preimmune serum IgG. This indicated an increased susceptibility of Leishmania promastigotes to CML upon incubation with anti-fusion protein IgG.

Promastigotes pre-treated with purified anti-fusion protein IgGs, were incubated with BALB/c peritoneal macrophages. A drastic drop in the percentage of bound promastigotes could be observed with increasing amount of IgG used, thus indicating the ability of anti-fusion protein IgGs to block promastigote-macrophage binding as compared to untreated promastigotes or promastigotes incubated with preimmune serum IgG.