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 Methanosarcina acetivorans was developed which uses the highly regulated cdh promoter and allows expression of recombinant protein with optional 6xHis protein fusions to facilitate rapid purification. A protocol for high-density mass cultivation of M. acetivorans in a stainless steel bioreactor configured as a pH-auxostat was developed. The cdh promoter and alternate promoters were analyzed in attempt to enhance expression of recombinant proteins. The protein expression system was tested on several proteins: the Methanocaldococcus jannaschii prolyl tRNA synthetase, the M. acetivorans prolyl tRNA synthetase, the Methanosarcina thermophila carbonic anhydrase, and the Dehalococcoides ethenogenes tricholorethylene dehalogenase.