[Construction and expression of a fusion protein containing extracellular domain of human Jagged1 and Fc fragment of human IgG1 in Pichia Pastoris].
In order to construct a pichia pastoris expression vector containing the extracellular domain of human Jagged1 and the Fc fragment of human IgG1 fusion gene, or containing only the Fc fragment of human IgG1 and to express them in pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into pIC-Fc vector constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by means of electroporation. The recombinant transformants with a high copy number of the plasmid were selected on MD plate with G418. The expression of protein was induced by addition of methanol. Then, protein expression was analyzed by SDS-PAGE. The results indicated that the extracellular domain of human Jagged1 gene was effectively amplified. The DNA sequencing result showed that the constructed plasmid containing hJagged1(ext)-Fc fusion gene was the same as designed. The fusion protein was successfully expressed in Pichia pastoris. It is concluded that the hJagged1(ext) gene has been successfully cloned and expressed, which provides a new fusion protein for further experiments, the hJagged1(ext)-Fc fusion protein can be used as a new stimulator for proliferation of hematopoietic stem/progenitor cells in vitro.
Li
Guo-Hui
GH
Department of Hematology, Tangdu Hospital, The Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China.
Kang
Zhi-Jie
ZJ
Huang
Si-Yong
SY
He
Fei
F
Xu
Heng
H
Zhang
Li
L
Wu
Yan-Lan
YL
Niu
Xiao-Li
XL
Ma
Chang-Sheng
CS
Han
Hua
H
Liang
Ying-Min
YM
D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis* / Q000235:genetics; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D000072099:Serrate-Jagged Proteins; D014162:Transfection
D002135:Calcium-Binding Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000072099:Serrate-Jagged Proteins