Cloning, expression, and characterization of an acetolactate synthase (ALS) gene from Anabaena azotica

Abstract

A novel acetolactate synthase (ALS) gene was cloned from Anabaena azotica, which was isolated from China and is regarded as a vital and beneficial photosynthetic microorganism that can contribute to soil fertility and maintain ecosystem stability. The full-length gene consists of an open reading frame (ORF) of 1644bp and encodes a 59.2kDa 547 amino acid protein. Sequence alignment using the BLAST similarity search in the GenBank database revealed that the amino acid sequence was similar to ALS derived from Anabaena sp. PCC 7120. The ALS gene was subcloned into the plasmid pET28a (+) and expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant protein was purified and subsequently characterized. The properties of ALS from the E. coli transformant are similar to that of an enzyme from the original A. azotica. Conserved amino acid sequence and the predicted structure of the active site were consistent with those observed for the bacterial enzyme Klebsiella pneumoniae ALS in similar experiments. Our results represent a significant functional demonstration of the existence of an algae ALS subunit and provide a solid basis for further studies on the structure, function and properties of ALS, as well as its response mechanism to inhibitors.