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PROTEIN EXPRESSION AND PURFICATION OF ADENYLATE KINASE from Methanococcus Maripaludis
Author(s) -
Valencia Valerie,
Counago Rafael,
Shamoo Yousif
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a986-d
Subject(s) - methanococcus , adenylate kinase , gene , hyperthermophile , biology , chemistry , biochemistry , enzyme , archaea
Adenylate kinase (ADK ‐ E.C. 2.4.3.7) catalyzes the interconversion of adenylate species (AMP, ADP, ATP), maintaining their homeostasis in the cell. ADK’s key function in energy metabolism makes it a ubiquitous enzyme, found in all domains of life. ADK’s isolated from archaeabacteria are of special interest to the study of protein evolution due to their peculiar trimeric form and unusual temperature stability. Here we study the trimeric ADK from Methanococcus maripaludis , a mesophilic methanogenic archaeabacterium. We have cloned the gene for M. maripaludis trimeric ADK in an E. coli protein expression vector (pET11a). However, attempts to express M. maripaludis ADK in E. coli BL21 (De3) cells have not been successful. Due to its archaeal origin, M. maripaludis ADK gene employs codons that are unusual to eubacterial organisms, such as E. coli . Therefore, we have also attempted to express the archaeal enzyme in a special E. coli strain (Rosetta) that produces the t‐RNAs for the rare codons found in the archaeal gene. Overexpression of M. maripaludis ADK in Rosetta cells was successful. We have also shown that M. maripaludis ADK can complement for the enzyme deficiency in a temperature‐sensitive strain of E. coli (CV‐2). We are currently varying protein expression conditions (temperature and inducers concentrations) in order to optimize M. maripaludis expression in E. coli . The overexpressed protein will be purified and used for biochemical and biophysical studies aimed at understanding the evolution of protein stability in the ADK family of enzymes.