[Expression of glutathione-S-transferase fusion protein and human CCL3L1 protein].
OBJECTIVE
To clone human CCL3L1 cDNA and to express and purify the glutathione-S-transferase (GST) fusion protein and human CCL3L1 protein.


METHODS
Total 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.


RESULTS
As 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.


CONCLUSIONS
GST-CCL3L1 fusion protein can be successfully expressed using appropriate vector.
Xu
Bin
B
Department of Infectious Diseases, Beijing Youan Hospital, Capital University of Medical Sciences, Beijing 100069, China.
Shi
Ying
Y
Li
Jun-Hong
JH
Zhang
Wei
W
Wu
Hao
H
Chen
De-xi
DX
D045744:Cell Line, Tumor; D019742:Chemokines, CC / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
C492767:CCL3L1 protein, human; D019742:Chemokines, CC; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase