Vaccinia virus-mediated high level expression and single step purification of recombinant Jak2 protein.
Jak2 functions as a non-receptor tyrosine kinase and has been linked to pathologies such as cancer and cardiovascular disease. Because of this, many studies have tried to better understand its function. Unfortunately, very little information is known about its catalytic or biochemical properties as purification of significant amounts of functional Jak2 protein has been exceedingly difficult. Here, Jak2 was expressed in BSC-40 cells using a vaccinia virus-mediated expression system. Significant amounts ( approximately 10microg) of Jak2 protein were expressed from a single 100-mm cell culture dish. The protein was first harvested using three different methods of extraction to determine the relative efficiency of each lysis method with respect to Jak2 protein yield and catalytic activity. We found that lysis methods utilizing detergents increased the efficiency of protein extraction about 3-fold when compared to a method lacking detergent. However, with respect to catalytic activity, Jak2 isolated from cells using detergent-containing lysis buffers had significantly less catalytic activity than when compared to the method that was detergent free. Expression was then scaled up and Jak2 protein was purified via a one step immunoaffinity purification scheme using both the detergent-free and a modified detergent-containing method of extraction that maintained catalytic activity. In vitro kinase assays demonstrated that the purified product was highly catalytic as measured by its ability to tyrosine phosphorylate Stat1. Collectively, the results show that (1) Jak2 can be expressed at very high levels in mammalian cells, (2) it can be purified to homogeneity via a single step purification scheme, and (3) the purified product is biologically active.
Ma
Xianyue
X
Department of Physiology and Functional Genomics, University of Florida College of Medicine, P.O. Box 100274, Gainesville, FL 32610, USA.
Sayeski
Peter P
PP
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers; D053614:Janus Kinase 2; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014616:Vaccinia virus / Q000235:genetics*
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D053614:Janus Kinase 2