Expression of glutathione-S-transferase fusion protein and human CCL3L1 protein.
- Author:
Bin XU
1
;
Ying SHI
;
Jun-Hong LI
;
Wei ZHANG
;
Hao WU
;
De-xi CHEN
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line, Tumor; Chemokines, CC; biosynthesis; genetics; Cloning, Molecular; DNA, Complementary; genetics; Escherichia coli; drug effects; genetics; metabolism; Female; Genetic Vectors; Glutathione Transferase; biosynthesis; genetics; Humans; Isopropyl Thiogalactoside; pharmacology; Recombinant Fusion Proteins; biosynthesis; Reverse Transcriptase Polymerase Chain Reaction
- From: Acta Academiae Medicinae Sinicae 2006;28(5):642-646
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo clone human CCL3L1 cDNA and to express and purify the glutathione-S-transferase (GST) fusion protein and human CCL3L1 protein.
METHODSTotal RNA was isolated from breast cancer cell line MCF7. CCL3L1 cDNA including open reading frame was obtained by RT-PCR. PCR product was digested with EcoR I and cloned into the pGEX-4T-1 vector. The plasmids from positive clone was prepared and sequenced to confirm the CCL3L1 in correct fusion form. pGEX-4T-CCL3L1 was transfected to BL21 E. coli via isopropyl-beta-D-thiogalactoside (IPTG) induction to produce GST-CCL3L1 fusion protein, which was further detected by SDS-PAGE and Western blotting.
RESULTSAs shown and confirmed by restriction endonuclease digestion analysis, CCL3L1 was correctly inserted into pGEX-4T-1 vector. The expressed fusion protein had a relative molecular weight of approximately 34 kD.
CONCLUSIONGST-CCL3L1 fusion protein can be successfully expressed using appropriate vector.
