[Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor].
OBJECTIVE
To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF).


METHODS
In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay.


RESULTS
In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L.


CONCLUSIONS
c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.
Su
Lin
L
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005, China.
Liu
Chang-zheng
CZ
Deng
Yan-chun
YC
Yang
Ke-gong
KG
Liang
Zhi-quan
ZQ
Chen
Song-sen
SS
D002478:Cells, Cultured; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008024:Ligands; D010957:Plasmids; D019009:Proto-Oncogene Proteins c-kit / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
D007136:Immunoglobulins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D019009:Proto-Oncogene Proteins c-kit