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Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies

Nera, KP; Brockmann, E; Vihinen, Mauno LU ; Smith, CIE and Mattsson, PT (2000) In Protein Expression and Purification 20(3). p.365-371
Abstract
Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleck-strin 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... (More)
Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleck-strin 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. (C) 2000 Academic Press. (Less)
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author
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
20
issue
3
pages
365 - 371
publisher
Academic Press
external identifiers
  • wos:000165611300003
  • scopus:0033667659
ISSN
1046-5928
DOI
10.1006/prep.2000.1316
language
English
LU publication?
no
id
f6040da6-895b-4a4c-9dd4-44bec3eeade1 (old id 3851963)
date added to LUP
2013-06-28 15:28:50
date last changed
2017-01-01 04:57:50
@article{f6040da6-895b-4a4c-9dd4-44bec3eeade1,
  abstract     = {Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase consisting of N-terminal pleck-strin 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. (C) 2000 Academic Press.},
  author       = {Nera, KP and Brockmann, E and Vihinen, Mauno and Smith, CIE and Mattsson, PT},
  issn         = {1046-5928},
  language     = {eng},
  number       = {3},
  pages        = {365--371},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {Rational design and purification of human Bruton's tyrosine kinase SH3-SH2 protein for structure-function studies},
  url          = {http://dx.doi.org/10.1006/prep.2000.1316},
  volume       = {20},
  year         = {2000},
}