The mD plasmid containing CD

The mD1.2 plasmid containing CD4 domain 1 gene under the control of a promoter was obtained as a gift from Dr. Dimiter S. Dimitrov. The mD1.2 plasmid was digested using restriction endonuclease I (New England BioLabs, USA) to release the mD1.2 gene. The gene encoding the mD1.2 protein was elongated using the DNA polymerase (New England BioLabs, USA) and two synthesized oligonucleotides (IDT, USA): a forward oligonucleotide (5′-GGA TAT ACA TAT GAC CGT GGC CCA GGC GGC CAA GAA GGT GGT GTA CG-3′) containing restriction site for I (New England BioLabs, USA) at the N-terminus of the mD1.2 gene, and a reverse oligonucleotide (5′-CGC GGA TCC TTA CAT CAT GCC GTG ATG GTG GTG ATG GTG GCC GGC CTG GCC GCC TAC CAC TAC CAG-3′) containing a hexahistidine casette and a restriction site for HI (New England BioLabs, USA) at the C-terminus of the mD1.2 gene. After digestion using I and HI, the modified CD4 domain 1 (mD1) gene was inserted into a pET16 vector. Site-directed mutagenesis of position 28 of mD1 was carried out using a primer 5′-CAG AAG AAG AAC ATC CAG TTC CAC AAG AAC AGC AAC CAG ATC AAG ATC-3′ following a reported procedure. The acridon-2-ylalanyl-tRNA was prepared following our reported procedure. The in vitro expression mixture (600μL total volume) contained 60μg of modified mD1 (TAG at position 28) plasmid DNA, 300μL of premix (35mM Tris-acetate, pH 7.0, containing 190mM potassium glutamate, 30mM ammonium acetate, 2.0mM dithiothreitol, 11mM magnesium acetate, 20mM phospho(enol)pyruvate, 0.8mg/mL of tRNA, 0.8mM IPTG, 20mM ATP and GTP, 5mM CTP and UTP and 4mM cAMP), 100μM of each of the 20 amino acids, 60μCi of []--methionine, 10μg/μL rifampicin, 180μg of aminoacylated tRNA and 180μL of S-30 extract from strain BL21(DE3)., The reaction mixture was incubated at 30°C for 45min. Plasmid DNA containing the gene for wild-type mD1 was used as a positive control, and an abbreviated tRNA (tRNA-C) lacking any amino Cinobufagin was used as the negative control. An aliquot containing 2μL of reaction mixture was removed, treated with 2μL of loading buffer and heated at 90°C for 2min. The sample was analyzed by 15% SDS–PAGE at 100V for 2h. The wild-type and modified mD1s containing a C-terminal hexahistidine fusion peptide were purified by Ni–NTA chromatography. The in vitro translation reaction mixture (600μL) was diluted with 1800μL of 50mM Tris–HCl, pH 8.0, containing 300mM NaCl and 10mM imidazole, and mixed gently with 150μL of 50% slurry of Ni–NTA resin at 4°C for 1h. Then the mixture was applied to a column and washed with 800μL of 50mM Tris–HCl, pH 8.0, containing 300mM NaCl and 10mM imidazole. Finally, the mD1 protein was eluted four times with 150-μL portions of 50mM Tris–HCl, pH 8.0, containing 300mM NaCl and 150mM imidazole. Aliquots of each fraction were analyzed by 15% SDS–PAGE. The eluates containing mD1 protein were combined and loaded on a Sephadex-G25 column (10×0.5cm) and eluted ten times with 150μL of 50mM Tris–HCl, pH 8.0. Aliquots of each fraction were analyzed again by 15% SDS–PAGE. The fluorescence spectra of modified mD1 containing acridon-2-ylalanine at position 28 was measured using a Varian Cary Eclipse Fluorescence Spectrophotometer with the excitation slit set at 10nm and emission slit at 10nm. The emission spectra of modified mD1 protein sample was recorded at pH 8.0 following excitation at 260nm. To 50μL of 1.0μM modified mD1 protein sample was added 2.0–10.0μM Evans blue (EB) (MP Biomedicals, USA) or 0.2–1.0μM HIV-1 gp120 (Fitzgerald Industries International Inc, USA). After incubation at room temperature for 10min, the emission spectra of modified mD1 protein samples were recorded following excitation at 260nm. Human membrane protein CD4 contains four immunoglobulin domains (D to D) that are found on the extracellular surface of immune cells such as T cells, macrophages, monocytes and dendritic cells. These four domains are responsible for binding to different regulatory molecules in human blood. For HIV-1 infection, the binding of domain 1 (D1) of CD4 on T cells to HIV-1 gp120 protein initiates viral entry. Recently, Chen et al. have designed and screened several D1 mutant libraries. They found a mutant CD4 domain 1 (mD1.2) protein having better solubility (2-fold) and much stronger binding ability to HIV-1 gp120 (50-fold) than the soluble CD4 (sCD4) receptor. The properties of this mD1.2 protein were used to develop a fluorescent probe to detect the HIV-1 gp120 protein in this study.