Expression of full-length human alkylglycerol monooxygenase and fragments in Escherichia coli
| Autor: | Mayer Matthias, Keller Markus A., Watschinger Katrin, Werner-Felmayer Gabriele, Werner Ernst R., Golderer Georg |
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| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: | |
| Zdroj: | Pteridines, Vol 24, Iss 1, Pp 111-115 (2013) |
| Druh dokumentu: | article |
| ISSN: | 0933-4807 2195-4720 |
| DOI: | 10.1515/pterid-2013-0014 |
| Popis: | Alkylglycerol monooxygenase (AGMO; EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl ether bond of alkylglycerols in humans. It is an integral membrane protein with nine predicted transmembrane domains. We attempted to express and purify full-length and truncated forms of AGMO in Escherichia coli. Full-length AGMO could not be expressed in three different E. coli expression strains, three different expression vectors and several induction systems. We succeeded, however, in expression of three N-terminally strep-tagged truncated forms, named active sites 1, 2 and 3, with 205, 134 and 61 amino acids, respectively. Active site 1 fragment, containing two predicted transmembrane regions, a membrane associated region and all known amino acid residues important for catalytic activity, was not fully soluble even in 8 M urea. Active site 2 containing only one predicted membrane associated domain required 8 M urea for solubilisation and eluted in gel filtration in 1 M urea as a trimer. Active site 3 with no hydrophobic domain eluted in gel filtration in 1 M urea as monomer and dimer. These results show that even truncated forms of AGMO are barely soluble when expressed in E. coli and show a high tendency for aggregation. |
| Databáze: | Directory of Open Access Journals |
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