Production, purification and characterization of non-myristylated human T-cell protein tyrosine kinase in a baculovirus expression system.
A non-myristylated form (LCK M) of the human T-lymphocyte-specific protein tyrosine kinase (LCK) was produced at high levels in a baculovirus expression system (BVES) using two strategies. First, LCK M was produced by direct expression of a Gly2 --> Ala mutant of LCK. Second, LCK was produced as a glutathione S-transferase (GST) fusion, and LCK M was derived from the fusion protein by cleavage with thrombin. Both recombinant proteins (re-proteins) were produced at 5% of the total protein of infected Spodoptera frugiperda (Sf9) cells and were purified to >95% homogeneity. The enzymatic properties of the re-proteins and their inhibition by protein kinase inhibitors were comparable to the native enzyme (LCK N) derived from Jurkat cells and wild-type LCK derived from the BVES. The high production levels will facilitate the recovery of large quantities of re-protein for use in biochemical and biophysical studies.
Lehr
R V
RV
Department of Molecular and Cellular Biology, Sterling Winthrop Inc., Collegeville, PA 19426-0900, USA.
Ma
Y G
YG
Kratz
D
D
Brake
P G
PG
Wang
S
S
Faltynek
C R
CR
Wang
X M
XM
Stevis
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013601:T-Lymphocytes / Q000201:enzymology*; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases