Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes
PROTEIN EXPRESSION AND PURIFICATION, cilt.73, ss.223-230, 2010 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 73
- Basım Tarihi: 2010
- Doi Numarası: 10.1016/j.pep.2010.05.004
- Dergi Adı: PROTEIN EXPRESSION AND PURIFICATION
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
- Sayfa Sayıları: ss.223-230
- Orta Doğu Teknik Üniversitesi Adresli: Evet
Özet
In this study we describe, the construction of a co-expression vector allowing simultaneous production of Thermoplasma volcanium 20S proteasome alpha- and beta-subunits in Escherichia coli. This heterologous expression system provided high level production of fully active 205 proteasome that can be purified easily by using a conventional two-step chromatographic technique. The recombinant proteasome was purified to homogeneity 12-fold with a specific activity of 26.5 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two unique bands of alpha-(24 kDa) and beta-(21 kDa) subunits which were combined into proteolytically active proteasome complex in vivo when they were co-expressed in E. coli. The predominant peptide hydrolyzing activity was measured with typical chromogenic substrate (Ala-Ala-Phe-pNA) for chymotrypsin-like activity. The sequence analyses of the subunit genes showed that functional domains and residues including catalytic groups are highly conserved as compared to other archaeal proteasomes.