Methanosarcina mazei is one of the model organisms for the methanogenic order Methanosarcinales whose metabolism has been\r\nstudied in detail. However, the genetic toolbox is still limited. This study was aimed at widening the scope of utilizable methods\r\nin this group of organisms. (i) Proteins specific to methanogens are oftentimes difficult to produce in E. coli. However, a protein\r\nproduction system is not available for methanogens. Here we present an inducible system to produce Strep-tagged proteins in Ms.\r\nmazei. The promoter p1687, which directs the transcription of methyl transferases that demethylate methylamines, was cloned\r\ninto plasmid pWM321 and its activity was determined by monitoring �Ÿ-glucuronidase production. The promoter was inactive\r\nduring growth on methanol but was rapidly activated when trimethylamine was added to the medium. The gene encoding the\r\n�Ÿ-glucuronidase from E. coli was fused to a Strep-tag and was cloned downstream of the p1687 promoter. The protein was\r\noverproduced in Ms. mazei and was purified in an active form by affinity chromatography. (ii) Puromycin is currently the only\r\nantibiotic used as a selectable marker in Ms. mazei and its relatives. We established neomycin resistance as a second selectable\r\nmarker by designing a plasmid that confers neomycin resistance in Ms. mazei.
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