Human recombinant laminin-binding protein: isolation, purification, and crystallization.
The mRNA of the precursor of laminin-binding protein (LBP) was isolated from a human embryo kidney cell line and cloned. The determined sequence of the LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cells, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE was 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool against LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombinant protein. For this purpose, crystals of the human LBP were obtained by the standing drop version of the pore diffusion technique. The crystals appropriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 A.
Sorokin
A V
AV
Institute of Molecular Biology, State Research Center for Virology and Biotechnology "Vector", Koltsovo, Novosibirsk Region, Russia.
Mikhailov
A M
AM
Kachko
A V
AV
Protopopova
E V
EV
Konovalova
S N
SN
Andrianova
M E
ME
Netesov
S V
SV
Kornev
A N
AN
Loktev
V B
VB
D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000737:chemistry; D011485:Protein Binding; D017474:Receptors, Laminin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism
D017931:DNA Primers; D017474:Receptors, Laminin; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C065696:glycoprotein E, Flavivirus