Overexpression of the Thermus aquaticus B malate dehydrogenase-encoding gene in Escherichia coli.
Expression of the Thermus aquaticus B malate dehydrogenase (MDH)-encoding gene (mdh), cloned in Escherichia coli, was initially at a relatively low level (0.1% of soluble cell protein) and was effected by read-through from the tac promoter in the plasmid vector used. An enhancement in expression to 0.4% of soluble cell protein was achieved by shortening the intervening sequence between the promoter and the translation start codon of mdh. An NdeI restriction site (5'-CAT-ATG-3') was engineered in the shortened fragment, which also changed the start codon from GTG to ATG. This resulted in an eightfold increase in expression, to 3.2% of soluble cell protein. Expression was further increased by subcloning the mdh gene via the engineered NdeI site, into two plasmid expression vectors, one carrying the E. coli trpP promoter and the other the E. coli mdhP promoter. In both these expression systems, 40-50% of the soluble cell protein was T. aquaticus MDH. This suggests that expression of the cloned T. aquaticus mdh in E. coli is enhanced predominantly by the optimisation of transcription and translation initiation signals. Moreover, the base composition of the coding region and the pattern of codon usage dictated by it appear to have little effect on expression. Heat treatment of the cell extract at 85 degrees C further effected purification of T. aquaticus MDH to over 80% of the soluble cell protein. The MDHs purified to homogeneity from the high-expression clones were identical with the MDH isolated from T. aquaticus B cells with respect to all measured parameters.
Alldread
R M
RM
Division of Biotechnology, Centre for Applied Microbiology and Research, Porton, Salisbury, UK.
Nicholls
D J
DJ
Sundaram
T K
TK
Scawen
M D
MD
Atkinson
T
T
D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013696:Temperature; D013824:Thermus / Q000201:enzymology* / Q000235:genetics
D004274:DNA, Recombinant; D008291:Malate Dehydrogenase