Characterization of neuronal Src kinase purified from a bacterial expression system.
Neuronal Src (n-Src) is an alternative isoform of Src kinase containing a 6-amino acid insert in the SH3 domain that is highly expressed in neurons of the central nervous system (CNS). To investigate the function of n-Src, wild-type n-Src, constitutively active n-Src in which the C-tail tyrosine 535 was mutated to phenylalanine (n-Src/Y535F) and inactive n-Src in which the lysine 303 was mutated to arginine in addition to the mutation of Y535F (n-Src/K303R/Y535F), were expressed and purified from Escherichia coli BL21(DE3) cells. We found that all three types of n-Src constructs expressed at very high yields (∼500 mg/L) at 37°C, but formed inclusion bodies. In the presence of 8M urea these proteins could be solubilized, purified under denaturing conditions, and subsequently refolded in the presence of arginine (0.5M). These Src proteins were enzymatically active except for the n-Src/K303R/Y535F mutant. n-Src proteins expressed at 18°C were soluble, albeit at lower yields (∼10-20 mg/L). The lowest yields were for n-Src/Y535F (∼10 mg/L) and the highest for n-Src/K303R/Y535F (∼20 mg/L). We characterized the purified n-Src proteins expressed at 18°C. We found that altering n-Src enzyme activity either pharmacologically (e.g., application of ATP or a Src inhibitor) or genetically (mutation of Y535 or K303) was consistently associated with changes in n-Src stability: an increase in n-Src activity was coupled with a decrease in n-Src stability and vice versa. These findings, therefore, indicate that n-Src activity and stability are interdependent. Finally, the successful production of functionally active n-Src in this study indicates that the bacterial expression system may be a useful protein source in future investigations of n-Src regulation and function.
Marin
Vedrana
V
Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306-4300, USA.
Groveman
Bradley R
BR
Qiao
Haifa
H
Xu
Jindong
J
Ali
Mohammad K
MK
Fang
Xiao-Qian
XQ
Lin
Shuang-Xiu
SX
Rizkallah
Raed
R
Hurt
Myra H
MH
Bienkiewicz
Ewa A
EA
Yu
Xian-Min
XM
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
D007527:Isoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D019061:src-Family Kinases