Construction of Mycobacterial Expression Vectors
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Date
1999-06
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Addis Ababa,University
Abstract
Molecular biology studies on M. feprae and M. tllberculosis involve cloning
of genes and high level of expression to provide large amounts of protein.
These proteins are used 10 study their usefulness in sub-unit vacci nes or as
antigens in diagnostic kits. Most of the work that has been done to obtain
protein antigens directly from the pathogens has drawbacks. For example
the tedious purification methods and the inability to culture M.leprae in
vitro. Most of these obstacles can be circumvented by over-expressing
proteins in E.coli but that has its drawbacks as well. The posHransiationai
modification in £.coli is absent or at least different from that m
mycobacteria. Furthcnnore, some mycobacterial proteins can not be
produced in E.coli. We have init iated studies to develop a mycobacterial
expression vector th at might sidestep somc or the above namcd difficulties.
This study describes the construction or a vector that allows the overexpression
or mycobacterial proteins in a non-pathogenic, fast growing
mycobacterial host. Firstly, we have cloned a number of model proteins in
an E.coli expression system. These vectors allow the over-expression of
M.leprae and M.lllberclIlosis recombinant proteins (45kD, Esat-6 and Trx)
in E. coli. Secondly, a mycobacteri al expression vector (pDKl) was
constructed. This vector has a hi stidine-tag that can be used for affinity
purification or proteins. In this way we circumvent tedious biochemical
purification systems. It also contains a multiple cloning site for convenient
cloning of genes or interest. Thirdly, this pDK I expression vector was used
to introduce the same test proteins as for the E.coli expression vector. These
test proteins were over-expressed in E. coli as well as in a mycobacterial
host. Both protein sets can be used to detcnnine whether there is any
difference in recombinant prote ins obtained from E.coli and the semiautologous
system using M. smegmaris as a recombinant host.
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Construction of Mycobacterial Expression vectors