Molecular cloning and co-expression of Thermoplasma volcanium proteasome subunit genes.
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 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 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. 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.
Kocabiyik
Semra
S
Department of Biological Sciences, and Biomedical Engineering Graduate Program, Middle East Technical University, 06531 Ankara, Turkey. biosemra@metu.edu.tr
Ozdemir
Inci
I
Zwickl
Peter
P
Ozdoğan
Seda
S
D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004151:Dipeptides / Q000032:analysis / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D013379:Substrate Specificity
D019843:Archaeal Proteins; D004151:Dipeptides; D010455:Peptides; D011994:Recombinant Proteins; C014309:alanylphenylalanine; D010450:Endopeptidases; C093138:proteasome, Thermoplasma