[Construction of prokaryotic expression vectors of HAI-1 with different domains and expression of the fusion proteins].
OBJECTIVE
To construct prokaryotic expression vectors for different domains of hepatocyte growth factor activator inhibitor type 1(HAI-1) gene and express the fusion proteins for further investigation into their biological functions.


METHODS
Five pairs of primers were designed to amplify different cDNA fragments of HAI-1 (KD1, KD1+LDLR, KD2, LDLR+KD2 and KD1+LDLR+KD2). The PCR products were cloned into pGEM-T Easy vectors and sequenced. The cDNA fragments were subcloned into pIVEX2.3-MCS, a prokaryotic expression vector with 6xhis tag and transformed into E.coli BL21 (DE3). The expression of histidine-tagged fusion proteins were induced with IPTG and confirmed by Western blot. The histidine-tagged fusion protein KD1+LDLR+KD2 was purified through immobilized Ni(2+) absorption chromatographic column.


RESULTS
Different domains of HAI-1 with 6 x his tag were successfully expressed in E.coli BL21 (DE3). The expressed KD1+LDLR+KD2 domain with the relative molecular mass size of 22,200 was purified specifically through affinity chromatographic column and confirmed by Western blot.


CONCLUSIONS
Recombinant plasmid pIVEX2.3-MCS/HAI-1 can be expressed in E.coli BL21(DE3) and the purified fusion protein KD1+LDLR+KD2 can be used for further research on biological functions of different domains of HAI-1.
Zeng
Lei
L
Department of Emergency Medicine, 1st Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China. lleizeng@yahoo.com.cn
Cao
Jiang
J
D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011387:Prokaryotic Cells / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology*; D053491:Proteinase Inhibitory Proteins, Secretory / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D053491:Proteinase Inhibitory Proteins, Secretory; D011993:Recombinant Fusion Proteins; C109149:SPINT1 protein, human