Expression of the protein-tyrosine kinase p56lck by the pTRX vector yields a highly soluble protein recovered by mild sonication.
We have expressed the protein-tyrosine kinase p56lck as a fusion with glutathione-S-transferase (GST) or thioredoxin (TRX). While the GST-Lck fusion protein was found to be poorly soluble and solubilization techniques led to a high degree of degradation, the TRX-Lck fusion protein was found to be highly soluble. This solubilization was achieved by mild sonication in a simple low ionic strength buffer which makes the fusion protein immediately available for further use. Even in the absence of protease inhibitors, the TRX-Lck fusion protein exhibited no trace of degradation. We produced 40-60 mg of TRX-Lck/l of culture which was efficiently cleaved at the endopeptidase site. The TRX-Lck and GST-Lck fusion proteins exhibited similar phosphorylation activity.
Hlavac
F
F
INSERM U-152, ICGM Bâtiment G. Roussy, Paris, France.
Rouer
E
E
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)