Cloning and expressing of rubredoxin oxidoreductase from Clostridium acetobutylicum in Escherichia coli
Rudolph, Frederick B.
Master of Arts
A 550 bp DNA fragment which apparently contains part of the rubredoxin oxidoreductase gene has been amplified by a polymerase chain reaction employing genonic DNA as template. Oligonucleotides used in this amplification were designed based on the multiple alignment of rubredoxin oxidoreductase from related species. An EMBL3 $\lambda$ phage library of C. acetobutylicum genomic DNA was screened by an oligonucleotide hybridization method. The phages which hybridized with the radiolabeled PCR product were subcloned into pUC19 and pACYC184 vectors, with the recombinant plasmids being selected on the basis of white/blue color screening method and the insertional inactivation method, respectively. The probable identity of the pUC19 clone, which was designated RuNC51 and carried a 3.0 kb BglII fragment, was confirmed by rubredoxin oxidoreductase enzyme activity assay. The sequence of RuNC51 showed the same level of similarity to related genes. It also surprisingly showed a 53% similarity to PriA (protein n$\sp\prime$) which is involved in DNA replication.