Recombinant human CIS2 (SOCS2) protein: subcloning, expression, purification, and characterization.
The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS-PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Eva
E
Biener
Sarah
S
Maurice
Yael
Y
Sandowski
Yael
Y
Cohen
Eugene E
EE
Gusakowsky
Robert
R
Hooghe
Akihiko
A
Yoshimura
Oded
O
Livnah
Arieh
A
Gertler
D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies; D008958:Models, Molecular; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins; D013379:Substrate Specificity; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D011506:Proteins; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D012097:Repressor Proteins; C106643:SOCS2 protein, human; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators