Cloning, sequencing and expression in Escherichia coli of the rubredoxin gene from Clostridium pasteurianum

Abstract

A 3.9 kb BglII-HindIII DNA fragment containing the rubredoxin gene from Clostridium pasteurianum has been cloned using oligonucleotide probes designed from the protein sequence. The 2675 bp SspI-HindIII portion of this fragment has been sequenced and found to contain three open reading frames in addition to the rubredoxin gene. The putative product of one of these open reading frames is similar to various thioredoxin reductases. The rubredoxin gene translates into a sequence that differs from the previously published protein sequence in three positions, D-14, D-22 and E-48 being replaced by the corresponding amides. These changes have been confirmed by partial resequencing of the protein. Promoter-like sequences and a transcription termination signal have been found near the sequence of the rubredoxin gene, which may therefore constitute an independent transcriptional unit Expression of C. pasteurianum rubredoxin in Escherichia coli strain JM 109 has been optimized by subcloning a 476 bp SspI-SspI fragment encompassing the rubredoxin gene. Under these conditions, the latter gene was partly under the control of the lac promoter of pUC18, and the level of rubredoxin production could be increased twofold on addition of a lactose analogue, thus reaching 2-3 mg of pure protein/1 of culture. Recombinant rubredoxin was produced in E. coli cells as the holoprotein, and displayed a u.v.-visible-absorption spectrum identical with that of the rubredoxin purified from C. pasteurianum. M.s. and N-terminal sequencing showed that C. pasteurianum rubredoxin expressed in E. coli differs from its native counterpart by having an unblocked N-terminal methionine.