ISSN:
1432-072X
Keywords:
Key wordsChloroflexus aurantiacus
;
Green bacteria
;
Phototrophic bacteria
;
Malate dehydrogenase
;
Lactate
;
dehydrogenase
;
mdh
;
Gene expression
;
σ70 Promoter
;
Thermostable protein
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract The gene encoding malate dehydrogenase (MDH) from Chloroflexus aurantiacus was cloned, sequenced, and analyzed. The mdh gene corresponded to a polypeptide of 309 amino acids with a molecular mass of 32,717 Da. The primary structure and the coenzyme-binding domain showed a high degree of similarity to lactate dehydrogenase (LDH), whereas the conserved amino acids that participate in substrate binding were those typical of MDHs. Using PCR techniques, the mdh gene was cloned in the expression vector pET11a, and large amounts of active C. aurantiacus MDH were produced in Escherichia coli after induction with isopropyl β-d-thiogalactoside. The expressed enzyme thus obtained was purified and retained full activity at 55° C. High levels of expression of mdh were also observed when the gene and its flanking sequences were cloned into pUC18/19, indicating that the putative σ70 promoter sequences found upstream of the C. aurantiacus mdh functioned in E. coli. When these sequences were deleted, the expression in E. coli was reduced dramatically.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002030050337
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