High yield bacterial expression and purification of active recombinant PA28alphabeta complex.
The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon gamma inducible PA28alphabeta, made of the two related subunits PA28alpha and beta, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its beta subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28alpha and beta subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28alphabeta complexes, that remain soluble and can be easily purified by regular chromatographic procedures.
Le Feuvre
Aurélie Y
AY
CRBM-CNRS, UMR 5237, Universités Montpellier 1 and 2, 1919 route de Mende, 34293 Montpellier Cedex 05, France.
Dantas-Barbosa
Carmela
C
Baldin
Véronique
V
Coux
Olivier
O
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D046988:Proteasome Endopeptidase Complex / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D009124:Muscle Proteins; C085047:PSME1 protein, human; D021122:Protein Subunits; D011994:Recombinant Proteins; C486207:PSME2 protein, human; D046988:Proteasome Endopeptidase Complex