Overproduction of bacterial chaperones improves the solubility of recombinant protein tyrosine kinases in Escherichia coli.
The production and purification of recombinant proteins from E. coli expression systems is often complicated by the fact that a significant amount of the foreign protein is deposited in the cytoplasm of the bacteria as aggregates or inclusion bodies. Many non-receptor protein tyrosine kinases are typical examples of proteins that are poorly soluble when overproduced in E. coli. Here, we report on the engineering of bacterial strains which overproduce chaperone proteins of the Hsp70 (DnaK, DnaJ and GrpE) and Hsp60 (GroEL and GroES) families. The simultaneous overproduction in E. coli of the chaperones DnaK, DnaJ and GrpE on the one hand, or GroEL and GroES on the other hand, and the human non-receptor protein tyrosine kinases Csk, Fyn or Lck resulted in increased solubility of the recombinant kinases. This provides the basis for future successful production and purification of large quantities of soluble and active non-receptor protein tyrosine kinases from E.coli expression systems and will enable the further characterization of these important enzyme families at the molecular level.
Caspers
P
P
Department of Biology, F. Hoffmann-La Roche, Basel, Switzerland.
Stieger
M
M
Burn
P
P
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D000081247:CSK Tyrosine-Protein Kinase; D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D019061:src-Family Kinases
D001426:Bacterial Proteins; D018834:Chaperonin 60; D018840:HSP70 Heat-Shock Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C000631346:CSK protein, human