A gene coding for the flavodoxin from Clostridium MP was designed, synthesized, and expressed in Escherichia coli. The sequence of the coding region was derived from the published amino acid sequence of the protein (Tanaka, M., Haniu, M., Yasunobu, K.T., and Mayhew, S.G. (1974) J. Biol. Chem. 249, 4393-4397) and was designed for optimal expression and for use of the cassette mutagenesis approach. The structural gene was subassembled in three sections, each of which was constructed by the enzymatic ligation of three complementary pairs of chemically synthesized oligodeoxyribonucleotides having short single-stranded ends complementary to that of the adjacent pair. Colligation of the three sections produced the final structural gene which consists of 420 nucleotides. The synthetic gene was cloned behind the hybrid tac promoter (Amman, E., Brosius, J., and Ptashne, M. (1983) Gene (Amst.) 25, 167-178) in the pKK223-3 vector or adjacent to the strong T7 RNA polymerase promoter in the pET-3a expression vector (Rosenberg. A.H., Lade, B.N., Chui, D-S., Lin, S-W., Dunn J.J., Studier, F.W. (1987) Gene (Amst.) 56, 125-135) for expression in E. coli. Upon induction with isopropyl-β-D-thiogalactoside, the flavodoxin polypeptide was expressed from the artificial gene to levels approaching 20% of total extractable proteins using either expression system. The flavodoxin was purified from cellular extracts as the holoprotein containing bound flavin mononucleotide. The recombinant flavodoxin protein was found to have an ultraviolet/visible spectrum, amino-terminal sequence, and amino acid composition identical to the wild-type flavodoxin protein purified from Clostridium MP. This work represents the first chemical synthesis and expression in E. coli of an artificial gene coding for a bacterial flavodoxin.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1989|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology