Expression, purification, and solution structural studies of the Src family tyrosine kinase Lyn and the Myc family transcription factor B-myc
Abstract
Src tyrosine kinases require the chaperone proteins Hsp70, Hsp90, Cdc37, and others in order to properly fold. We were able to obtain low levels of the folded, active kinase domain of Lyn tyrosine kinase (kLyn) in E. coli, even though bacteria do not express many of these chaperones. Alternate tags, co-expression with Gro-ESL, and induction of endogenous heat shock proteins did not increase the level of soluble active kinase expressed in E. coli. Due to extreme toxicity in yeast, expression of kLyn in S. cerevisiae as well as P. pastoris leads to cell death within four hours of induction, and therefore intracellular yeast expression of active kinase is severely limited. To overcome this toxicity, a secreted form of the kinase as well as a kinase inactive mutant (K295M) was produced. Despite the presence of the requisite chaperones in yeast, secreted kLyn was insoluble and intracellular expression of kLyn-K295M in P. pastoris was similar to that of E. coli. B-myc is an endogenous N-terminal homologue of the transcription factor c-myc but lacks the C-terminal DNA binding region. The N-terminal region of myc family members is responsible for mediating protein-protein interactions that regulate transcription. In order to better understand the role that the N-terminus of myc proteins plays in the regulation of gene transcription, we have investigated the dynamics of B-myc using multidimensional NMR spectroscopy, free as well as bound to the transactivation inhibitor MM1 and the transactivation activator TBP. The narrow 1H-15N HSQC peak dispersion and the backbone amide relaxation properties of the protein characterize B-myc as unfolded in solution. In the presence of MM1, a small sub-set of the 1H-15N HSQC peaks from 15N-labeled B-myc shift, consistent with the MM1 binding region of B-myc. Peak shifts are also observed upon binding TBP that map to the C-terminal region of B-myc. Areas of significant change in the relaxation rates upon binding to MM1 are observed in the regions of the Myc Homology Boxes 1 and 2 (MBI and MBII).
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biochemistry
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