Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies.
Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleckstrin homology (PH) domain followed by Tec homology (TH) domain, Src homology 3 and 2 (SH3 and SH2) domains, and a C-terminal kinase domain. Mutations in the human BTK gene cause the severe immunodeficiency disease X-linked agammaglobulinemia (XLA). The structural and functional basis of several XLA-causing mutations remains unknown, since only the structures of the PH and SH3 domains of human Btk are currently available. In this study, we overexpressed and purified a protein consisting of the SH3 and SH2 domains of human Btk for biochemical and structural analysis. The purified protein was only partially soluble and had a tendency to dimerize, which made it unsuitable for further studies. To overcome the problems of low solubility and dimerization, subdomain interactions were engineered without altering the function of the protein.
Nera
K P
KP
Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
Brockmann
E
E
Vihinen
M
M
Smith
C I
CI
Mattsson
P T
PT
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility; D013329:Structure-Activity Relationship; D018909:src Homology Domains
D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human