[High level expression of 5-helix protein in HIV gp41 heptad repeat regions and its virus fusion-inhibiting activity].
The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.
Wang
Jiuqiang
J
Veterinary Academy of Shandong Agricultural University, Tai'an 271018, China.
Pan
Xuwen
X
Tien
Po
P
Liu
Sidang
S
D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics*; D010455:Peptides / Q000235:genetics; D011994:Recombinant Proteins; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014760:Viral Fusion Proteins / Q000235:genetics; D053586:Virus Internalization / Q000187:drug effects*
C420917:5-helix protein, synthetic; D002352:Carrier Proteins; D015700:HIV Envelope Protein gp41; D010455:Peptides; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins; C514787:gp41 protein, Human immunodeficiency virus 1