Expression of recombinant proteins in the methane-producing archaeon Methanosarcina acetivorans

Abstract

Recombinant protein expression is a necessary tool for the investigation of proteins in the post-genomic era. While many systems exist for recombinant protein expression in organisms of the eukaryotic and eubacterial domains, few to none are available in the Archaea. A recombinant protein expression system using the methanogenic archaeon <em>Methanosarcina acetivorans</em> was developed which uses the highly regulated <em>cdh</em> promoter and allows expression of recombinant protein with optional 6xHis protein fusions to facilitate rapid purification. A protocol for high-density mass cultivation of <em>M. acetivorans</em> in a stainless steel bioreactor configured as a pH-auxostat was developed. The <em>cdh</em> promoter and alternate promoters were analyzed in attempt to enhance expression of recombinant proteins. The protein expression system was tested on several proteins: the <em>Methanocaldococcus jannaschii</em> prolyl tRNA synthetase, the <em>M. acetivorans</em> prolyl tRNA synthetase, the <em>Methanosarcina thermophila</em> carbonic anhydrase, and the <em>Dehalococcoides ethenogenes</em> tricholorethylene dehalogenase.