Chimeric Glutathione S-Transferases Containing Inserts of Kininogen Peptides
POTENTIAL NOVEL PROTEIN THERAPEUTICS*
- Amber A. Bentley‡,1,
- Sergei M. Merkulov§,1,
- Yi Peng¶,1,
- Rita Rozmarynowycz‡,
- Xiaoping Qi§,
- Marianne Pusztai-Carey¶,
- William C. Merrick¶,
- Vivien C. Yee¶,
- Keith R. McCrae‖,2 and
- Anton A. Komar‡,3
- From the ‡Center for Gene Regulation in Health and Disease, Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115,
- the §Department of Physiology and Biophysics and
- ¶Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, and
- the ‖Taussig Cancer Institute and Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195
- ↵2 To whom correspondence may be addressed. Tel.: 216-445-7809; Fax: 216-444-9464; E-mail: mccraek{at}ccf.org.
- ↵3 To whom correspondence may be addressed. Tel.: 216-687-2516; Fax: 216-687-6972; E-mail: a.komar{at}csuohio.edu.
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↵1 These authors contributed equally to this work.
Abstract
The study of synthetic peptides corresponding to discrete regions of proteins has facilitated the understanding of protein structure-activity relationships. Short peptides can also be used as powerful therapeutic agents. However, in many instances, small peptides are prone to rapid degradation or aggregation and may lack the conformation required to mimic the functional motifs of the protein. For peptides to function as pharmacologically active agents, efficient production or expression, high solubility, and retention of biological activity through purification and storage steps are required. We report here the design, expression, and functional analysis of eight engineered GST proteins (denoted GSHKTs) in which peptides ranging in size from 8 to 16 amino acids and derived from human high molecular weight kininogen (HK) domain 5 were inserted into GST (between Gly-49 and Leu-50). Peptides derived from HK are known to inhibit cell proliferation, angiogenesis, and tumor metastasis, and the biological activity of the HK peptides was dramatically (>50-fold) enhanced following insertion into GST. GSHKTs are soluble and easily purified from Escherichia coli by affinity chromatography. Functionally, these hybrid proteins cause inhibition of endothelial cell proliferation. Crystallographic analysis of GSHKT10 and GSHKT13 (harboring 10- and 13-residue HK peptides, respectively) showed that the overall GST structure was not perturbed. These results suggest that the therapeutic efficacy of short peptides can be enhanced by insertion into larger proteins that are easily expressed and purified and that GST may potentially be used as such a carrier.
Footnotes
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants HL089796 (to K. R. M.), GM68079 (to W. C. M.), and DK075897 and GM61388 (to V. C. Y. and Y. P.). This work was supported by National American Heart Association Grant 0730120N (to A. A. K.).
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The atomic coordinates and structure factors (codes 4ECB and 4ECC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
- Received April 17, 2012.
- © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
