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Gene expression in prokaryotic system |
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Principal Investigator : Lalit C Garg Project Associates/Assistants Ph D Students Collaborators 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. A. Cloning and expression of certain regions
of functional significance of gp63 of Leishmania donovani in translational
fusion with LTB, and testing the immunogenecity of the fusion proteins Fine mapping of
Leishmania gp63 protein has shown conserved immunogenic epitopes carrying the
binding domain SRYD for parasite-macrophage binding or catalytic domain HEXXH
for proteinase activity required for the intracellular parasitism in the host
cells. The present study aims to clone and express the conserved functional
domains of gp63, in fusion with E.coli heat labile enterotoxin chain B and
test the fusion proteins in vitro, for their ability to disrupt the
host-parasite interactions. In order to check the
expression of catalytic domain of gp63 in fusion with LTB, clone pQltbCat was
induced with IPTG. The protein was expressed in bulk in response to 2mM IPTG
as inclusion bodies, which were purified by the standard method for Ni-NTA
affinity purification. Since both the fusion
proteins LTB-Bind and LTB-Cat were expressed in insoluble form in E.coli, they
were then taken to a secretory expression vector carrying the LTB signal
sequence by SacI-HindIII digestion. The plasmids designated as pMltbCat and
pMltbBind were confirmed by insert specific digestions. The fusion proteins
were expressed in large amounts in native condition in the periplasmic space
in E.coli and secreted into the medium in V.cholerae. The fusion proteins were
purified from the V.cholerae culture medium by phosphocellulose ion-exchange
chromatography. The purified fusion
proteins were characterized by SDS-PAGE analysis, immunoblotting with anti-LTB
and anti-gp63 antibodies, ability of the native fusion proteins to pentamerize
and bind to GM1 ganglioside receptor on sandwich ELISAs. The purified denatured or
native fusion proteins were then used to immunize NZW rabbits by the
subcutaneous route. Booster dose was given after appropriate time intervals
with fusion protein in IFA. The antisera for the fusion proteins collected
through retro-orbital bleeding were tested for their immunogenecity by
immunoblotting and ELISAs using purified fusion proteins, purified Leishmania
gp63 and synthetic peptides corresponding to the gp63 binding and catalytic
domains as antigens. The rabbit anti-fusion protein antiserum was able to pick
up the fusion protein, pure LTB, and purified Leishmania gp63 on immunoblots.
The peptides spanning the catalytic and binding domains could be picked up by
the antiserum in ELISAs. To determine if the secretory fusion protein would be
immunogenic by itself, immunization of rabbits was done with the fusion
protein without any adjuvant. Fairly good titers could be obtained though they
were low as compared to immunization with adjuvant. For in vitro biological assays, IgG was purified from
rabbit anti-fusion protein antisera through Protein-G sepharose affinity
column. Fab fragments were also isolated by controlled digestion of purified
IgGs with papain. 10 mg of IgG was digested with 1 mg of papain at 37°C for
8-10 h and reaction terminated with 75 mM iodoacetamide. Fab fragments were
purified from the digestion mixture through CM-sepharose column. The antisera
or the purified IgG and Fab were then tested for their effect on biological
functions of Leishmania in vitro. Protease activity of gp63 The proteolytic activity of promastigote surface
protease gp63, in situ, was demonstrated by incubating azocasein with 2x109
live promastigotes. The generation of acid soluble azocasein peptides
catalyzed by the protease was measured by monitoring the absorbance at 366 nm.
A steady increase in absorbance as a function of time shows the proteolytic
activity of the promastigote suspension. Glutaraldehyde fixed promastigotes
and cell supernatant were used as positive and negative controls,
respectively. Preincubation of promastigotes with IgG of anti-LTB-cat
(expressed as inclusion bodies) resulted in significant decrease in
proteolytic activity of gp63. However not much of effect could be seen with
anti-LTB-bind or anti-LTB-cat (expressed in native condition). Anti-LTB and
rabbit preimmune IgG were taken as controls. Complement mediated lysis Complement mediated lysis (CML) of Leishmania
promastigotes was assessed by incubating the promastigotes (1x108cells/ml)
with different concentrations of normal Guinea pig serum (NGPS). After
incubation at 37°C for 30 min, number of intact, living promastigotes were
determined. The percentage of viable promastigotes was shown to decrease with
increasing concentration of NGPS. Heat inactivated NGPS (at 56°C) was used as
a control. After preincubation of promastigotes with fusion protein IgGs, the
cells were incubated with 10% NGPS. A drastic reduction in the number of
viable cells from 97% (at 0.5 mg) to 72% (at 1 mg) could be seen with anti-LTB-bind
IgG (expressed as inclusion bodies in E.coli). A slight increase in CML and
decline in viable cells could be seen upon further increasing the
concentration of IgG to 2 mg. No change in percentage of viable cells could be
observed with anti-LTB-cat, anti-LTB or preimmune serum IgG. Parasite-macrophage
attachment Promastigotes were
incubated with or without different Fab fragments of fusion protein IgGs at 4°C
for 1h. They were then added to the wells containing J774A-1 macrophage cell
line at a ratio of 20 promastigotes/cell. At the end of 1.5 h incubation,
unbound parasites were removed by washing and cover slips containing adherent
macrophages were fixed in methanol. Cover slips were then stained with Giemsa
and the number of promastigotes attached to macrophages was counted. The
percentage of parasites bound remained unaffected in case of parasites
incubated with increasing concentrations of Fab fragments of LTB-cat IgG, LTB-bind
IgG (against secretory fusion protein) and Fab fragments of LTB and pre-immune
IgG. When promastigotes were incubated with Fab fragment of LTB-bind IgG
(against inclusion body protein), significant reduction in the number of
attached parasites was observed. In case of parasites incubated with Fab
fragment of LTB-cat IgG (against secretory protein) a slight but significant
decline in the percentage of attached parasites was observed. B.
Production of site directed mutants of heat labile enterotoxin B and their
structure-function analysis. The main objective of
this study is to examine the role of a1 helix of LTB on structure, function
and expression of LTB in E.coli and V. cholerae system. Introduction of mutation
at N-terminal a1 helix of heat labile enterotoxin chain B gene by site
directed mutagenesis We have earlier reported
that the N-terminal 6 amino acids of a1 helix are crucial for expression and
folding of heat labile enterotoxin chain B in E.coli and V.cholerae. To
identify the crucial amino acids important for the structure-function
relationship, amino acids from position 2-7 were sequentially deleted to
generate different mutants MutP, MutQ, MutS, MutI, MutT and MutE. In addition
to these, a construct MutTE was made in which two amino acids at position 6
and 7 were deleted. Two substitution mutants at amino acid position 7 were
also made (MutED and MutEG). The mutant genes were cloned in expression vector
at Sac I-Hind III site. Recombinant clones and mutations were confirmed by DNA
sequencing. Expression studies in E.coli and V.cholerae showed expression of
mutant protein MutP, MutQ, MutE7G and MutE7D in periplasmic space while all
other mutant proteins (MutS, MutI, MutT, MutE and MutTE) failed to express.
mRNA of all the mutants was detected by Northern blot analysis. The mRNA for
mutant proteins, whose expression was not detected in vivo, could be
successfully translated using in vitro translation system. This suggests that
the mutants that failed to express proteins were not due to faulty translation
but faster degradation. Thus, the amino acids at position 4-7 are crucial for
protein stability. Relative levels of expression of mutant protein in
periplasmic space of E.coli when compared with wild type LTB protein were 92%
for MutED, 72% for MutP, 66% for MutQ and 48% for MutEG protein. Cloning of A-subunit gene
at the N-terminus of mutant B-subunit genes Heat labile enterotoxin
is transcribed as single polycistronic mRNA. Both the subunits of toxin are
synthesized and exported to periplasm of E.coli where B subunit binds with
A-subunit to form holotoxin. To investigate whether the presence of A-subunit
could rescue degradation of mutant LTB protein, MluI-SacI digested product of
A-subunit gene was cloned at N-terminus of different mutant B-subunit genes.
Expression of all these constructs was studied in E.coli and V.cholerae.
A-subunit was being expressed in all the mutant clones, however, this could
not rescue the degradation of mutant B protein (MutS, MutI, MutT, MutE and
MutTE) both in E.coli and V.cholerae. Mutant B proteins (MutP, MutQ, MutEG and
MutED) which were getting expressed alone, could also be co-expressed
efficiently with A-subunit as holotoxin in both the systems. Purification and
characterization of mutant proteins Mutant proteins MutP,
MutQ, MutEG, MutED, AMutP, AMutQ, AMutEG and AMutED that were getting secreted
into V.cholerae supernatant, were purified by phosphocellulose column
chromatography. Purified mutant B proteins (MutP, MutQ, and MutED) were used
for reconstitution of mutant holotoxin by mixing with purified wild type
A-subunit. These holotoxins are being analyzed for their biological activity. Intended mutations in the
recombinant protein were confirmed by N-terminal protein sequencing. CD
spectra revealed an alteration in the secondary structure of mutant proteins.
The mutant proteins retained the ability to form pentamer, to bind with
A-subunit, and also with GM1 ganglioside receptor. Affinity binding of mutant
B subunit with GM1 ganglioside receptor in 10 mM phosphate buffer showed 20%
reduction in case of MutP protein and 50% in MutQ protein when compared with
wild type LTB. In 80 mM phosphate buffer saline MutE7D protein showed higher
stability. Publications Original
peer-reviewed articles 1. *Das P, Tiwari G, Jain S and Garg LC (2000)
Nucleotide sequence of river buffalo beta-casein cDNA. J Anim Sci 78:1390 (*in
press last year, since published). 2.
*Das P, Tiwari G, Jain S and Garg LC (2000) Nucleotide sequence of river
buffalo kappa-casein cDNA. J Anim Sci 78:1389 (*in press last year, since
published). |