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Characterization of neuronal Src kinase purified from a bacterial expression system.

Title: Characterization of neuronal Src kinase purified from a bacterial expression system.
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Name(s): Marin, Vedrana, author
Groveman, Bradley R, author
Qiao, Haifa, author
Xu, Jindong, author
Ali, Mohammad K, author
Fang, Xiao-Qian, author
Lin, Shuang-Xiu, author
Rizkallah, Raed, author
Hurt, Myra H, author
Bienkiewicz, Ewa A, author
Yu, Xian-Min, author
Type of Resource: text
Genre: Journal Article
Text
Date Issued: 2010-12-01
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: 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.
Identifier: FSU_pmch_20558296 (IID), 10.1016/j.pep.2010.06.004 (DOI), PMC2952679 (PMCID), 20558296 (RID), 20558296 (EID), S1046-5928(10)00179-8 (PII)
Grant Number: R01 NS053567, R01 NS053567-04
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2952679.
Subject(s): Adenosine Triphosphate/metabolism
Amino Acid Substitution
Animals
Escherichia coli/genetics
Isoenzymes/chemistry
Isoenzymes/genetics
Isoenzymes/isolation & purification
Mice
Point Mutation
Protein Folding
Recombinant Proteins/chemistry
Recombinant Proteins/genetics
Recombinant Proteins/isolation & purification
src-Family Kinases/chemistry
src-Family Kinases/genetics
src-Family Kinases/isolation & purification
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_pmch_20558296
Owner Institution: FSU
Is Part Of: Protein expression and purification.
1096-0279
Issue: iss. 2, vol. 74

Choose the citation style.
Marin, V., Groveman, B. R., Qiao, H., Xu, J., Ali, M. K., Fang, X. -Q., … Yu, X. -M. (2010). Characterization of neuronal Src kinase purified from a bacterial expression system. Protein Expression And Purification. Retrieved from http://purl.flvc.org/fsu/fd/FSU_pmch_20558296