Optimal expression condition of recombinant RAP.
In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a(+) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni(+) -nitrilotriacetic acid (Ni(+) -NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were significantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni(+) -NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.
Zhang
Jie
J
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. tjzj121@sina.com
Zhang
Hong
H
Bi
Hao
H
Liu
Zhiguo
Z
Guo
Jianli
J
Qu
Shen
S
D000818:Animals; D002460:Cell Line; D002701:Chloramphenicol / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D026563:LDL-Receptor Related Protein-Associated Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013997:Time Factors; D014170:Transformation, Genetic
D018076:DNA, Complementary; D026563:LDL-Receptor Related Protein-Associated Protein; D011506:Proteins; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D002701:Chloramphenicol; D005947:Glucose