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Human recombinant laminin-binding protein: isolation, purification, and crystallization
A V Sorokin1, A M Mikhailov, A V Kachko
1Institute of Molecular Biology, State Research Center for Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia.
Biochemistry. Biokhimiia
|June 13, 2000
Summary
Researchers cloned and expressed human laminin-binding protein (LBP) in E. coli. The recombinant LBP interacts with tick-borne encephalitis virus protein E and was crystallized for X-ray analysis.
Area of Science:
- Molecular Biology
- Structural Biology
- Virology
Background:
- Laminin-binding protein (LBP) plays a role in cell adhesion.
- LBP has potential interactions with viral proteins.
Purpose of the Study:
- To clone, express, and purify human LBP.
- To investigate the interaction of recombinant human LBP with tick-borne encephalitis (TBE) virus protein E.
- To obtain crystals of human LBP for structural analysis.
Main Methods:
- mRNA isolation and gene cloning from human kidney cells.
- Bacterial expression of recombinant human LBP in E. coli.
- Purification using Ni-NTA-Sepharose chromatography.
- Western blot and enzyme immunoassay for interaction studies.
- Crystallization via pore diffusion technique for X-ray analysis.
Main Results:
- Identical LBP gene sequence across different human tissues.
- Homogeneous recombinant human LBP (43 kD) expressed and purified.
- Recombinant LBP recognized by monoclonal antibodies.
- Evidence of conserved epitope binding to TBE virus protein E.
- Successful crystallization of human LBP for X-ray diffraction to 2.5 A.
Conclusions:
- The recombinant human LBP retains biological activity and interaction capabilities.
- The structural analysis of LBP is feasible, paving the way for understanding its spatial arrangement.