Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes

• Published in: Protein expression and purification Vol. 73; no. 2; pp. 223 - 230
• Main Authors: Kocabıyık, Semra, Özdemir, İnci, Zwickl, Peter, Özdoğan, Seda
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2010
• Subjects: 20S proteasome; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Cloning, Molecular; Dipeptides - analysis; Dipeptides - metabolism; Electrophoresis, Polyacrylamide Gel; Endopeptidases - chemistry; Endopeptidases - genetics; Endopeptidases - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genetic Vectors; Heterologous expression; Humans; Hydrolysis; Peptides - genetics; Peptides - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - ultrastructure; Substrate Specificity; Thermoplasma volcanium; Thermoplasma volcanium; 20S proteasome; Heterologous expression
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.05.004

In this study we describe, the construction of a co-expression vector allowing simultaneous production of
Thermoplasma volcanium 20S proteasome α- and β-subunits in
Escherichia coli. This heterologous expression system provided high level production of fully active 20S 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 α-(24
kDa) and β-(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.
Structural analysis by electron microscopy of negatively stained
T. volcanium 20S proteasome revealed a unique conformational architecture (
i.e. a tubular structure of four-stacked heptameric rings with a sevenfold symmetric top view) that is perfectly conserved from procaryotes to human.