High level soluble expression, one-step purification and characterization of HIV-1 p24 protein
- Baozhong Zhang1Email author,
- Dabin Liu1Email author,
- Zuoyi Bao1,
- Bin Chen1,
- Cun Li1,
- Huanhuan Jiang1,
- Xiaona Wang1,
- Zhiqiang Mi1,
- Xiaoping An1,
- Jun Lu2Email author and
- Yigang Tong1Email author
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 14 March 2011
Accepted: 22 June 2011
Published: 22 June 2011
P24 protein is the major core protein of HIV virus particle and has been suggested as a specific target for antiviral strategies. Recombinant p24 protein with natural antigenic activity would be useful for various studies, such as diagnostic reagents and multi-component HIV vaccine development. The aim of this study was to express and purify the p24 protein in soluble form in E.coli.
According to the sequence of the p24 gene, a pair of primers was designed, and the target sequence of 700 bp was amplified using PCR. The PCR product was cloned into pQE30 vector, generating the recombinant plasmid pQE30-p24. SDS-PAGE analysis showed that the His-tagged recombinant p24 protein was highly expressed in soluble form after induction in E. coli strain BL21. The recombinant protein was purified by nickel affinity chromatography and used to react with HIV infected sera. The results showed that the recombinant p24 protein could specifically react with the HIV infected sera. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for the preparation of polyclonal antibody. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV p24 specific antibodies.
In this work, we report the high level soluble expression of HIV-1 p24 protein in E. coli. This soluble recombinant p24 protein specifically react with HIV infected sera and elicit HIV p24 specific antibodies in mice, indicating this soluble recombinant p24 protein could be a promising reagent for HIV diagnosis.
The human immunodeficiency virus type 1 (HIV-1) is the main cause of the acquired immunodeficiency syndrome (AIDS). Diagnosis of HIV infection, especially early diagnosis, is one of important part of AIDS prevention and control. Gag protein of HIV-1, a polyprotein of 55 kDa, is one of the most conserved viral proteins. The Gag protein is cleaved by a viral protease to release p17, p24 and p12 during viral maturation. P24 protein is the major core protein of the virus particle and has been suggested as a specific target for antiviral strategies. P24 protein is one of the detecting targets of most diagnostic kits. P24 antigen detection is also helpful for early diagnosis of HIV-infection. The fourth-generation test assays for HIV infection is established on the basis of the p24 antigen detection and is able to find the HIV-infected at an early stage, resulting in shortened diagnostic windows. The p24 protein also can be used as an integral part of any multi-component HIV vaccine[7, 8].
A proper recombinant p24 protein with the same antigentic activity as natural p24 protein would be useful for a number of studies. The p24 protein have been produced in a wide variety of systems, including Escherichia coli, Pichia pastoris, plant-based expression system[11, 12], baculovirus-insect cell, etc. In this study, a recombinant plasmid was constructed to express the His-tagged p24 protein in Escherichia coli. The protein was expressed in soluble forms and purified by Ni2+-NTA affinity chromatography. Enzyme-linked immunosorbant assay (ELISA) and Western blot analysis demonstrated that the recombinant p24 proteins exhibited good immunoreactivity and immunogenicity.
Strains, plasmids, enzymes and reagents
The E. coli strains DH5α and BL21(DE3) were used for cloning experiments and protein expressions, respectively. Both strains were purchased from Invitrogen (Novagen, Shanghai, China). Plasmid pQE30 (Novagen, Darmstadt, Germany) was used for recombinant protein expression. Restriction enzymes, Taq DNA polymerase, and T4 ligase were purchased from TaKaRa Biotechnology Co. (Dalian, China).
Construction of the plasmid expressing the p24 protein
The HIV-1 p24 open reading frame was amplified from plasmid pHIV which contains the HIV-1 NY5 and LAV strain hybrid genome  with the forward primer (5'-GAG GAT CCC CCA TAG TGC AGA ACC TC-3', BamHI site underlined), and the reverse primer (CCG GTA CCT TAG AAA ACT CTT GCT TTA TG-3', KpnI site underlined). The PCR product was digested with BamHI and KpnI and inserted into the prokaryotic expression pQE30 digested with the same enzymes to create the p24 expression plasmid pQE30-p24.
Expression of the p24 protein
E.coli BL21 transformed with pQE30-p24 was cultured in LB medium supplemented with 50 μg/ml ampicillin for growth at 37°C until the logarithmic phase (at OD600 of 0.5-0.6) and induced by isopropyl-β-D-Thiogalactoside (IPTG) at a final concentration of 1.0 mM for 12 h at 20°C. The bacterial lysates were subjected to 15% SDS-PAGE, and Bandscan5.0 software was applied to assess the expression of the fusion protein.
Characterization of the solubility of the p24 protein
To assess the solubility of the His-tagged p24 protein, logarithmic phase bacterial cultures were pelleted and suspended in 20 mM Tris-HCl lysis buffer (pH 8.0) supplemented with 100 mM NaCl, 1.0 mM phenylmethyl sulfonylfluoride (PMSF), 50 mg/ml lysozyme and subjected to sonication on ice until clear. The total bacterial proteins were then partitioned into soluble and insoluble fractions by centrifugation at 14,000 × g for 20 min at 4°C. The supernatant (soluble fraction) was collected and the pellets (insoluble fraction), which contained the inclusion bodies, were suspended in deionized water. Both fractions were analyzed in parallel by 15% SDS-PAGE to characterize the solubility of the His-tagged p24 protein.
Purification of the p24 protein
The supernatant was filtered through a 0.45-μm membrane (Pall Corporation, USA) and then loaded onto a gravity-flow column packed with 2 ml Ni2+-NTA resin slurry (Qiagen, Germany). His-tagged p24 fusion proteins were purified following the manufacturer's handbook for high-level expression and purification of 6×His-tagged protein and the yield was quantified using a Coomassie Protein Assay Kit (Biomed, China). 15% SDS-PAGE was performed to validate the identity and evaluate the purity of the target fusion protein.
Recombinant p24 protein identification
To confirm the presence and the apparent molecular mass of the recombinant proteins expressed in E. coli, Western blot was carried out using anti-His antibody (Sigma). The purified recombinant p24 protein were separated by 15% SDS-PAGE, electrotransferred onto a nitrocellulose membrane (GE Healthcare, USA) and blocked with 5% non-fat dry milk in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) at 37°C for 2 h. After washing 3 times (each 5 min) with TBS containing 0.05% Tween-20 (TBST), the membrane was incubated with horseradish peroxidase-conjugated anti-His monoclonal antibody. Immunoreactive proteins were then visualized using the ECL Western blotting analysis system (Pierce, Rockford, USA).
Immunoreactivity analysis of the recombinant p24 protein
Human serum samples (n = 90) were obtained from PLA Center for HIV Test, including forty HIV-1 positive samples and fifty HIV-1 negative human serum samples. The protein p24 (1.5 ug/ml in 200 nmol/L NaHCO3 pH9.8, 100 ul/well) were coated on ELISA plates (Nunc, Roskilde, Denmark) at 4°C overnight. Plates were then blocked at 37°C for 3 h with 5% non-fat milk and washed four times with PBST. Human sera were added as the primary antibody (1:50 dilution) at 37°C for 1 h. Plates were then washed four times with PBST and incubated with HRP-conjugated goat anti-human IgG (1:3000 dilution) at 37°C for 1 h. Color was developed using TMB solution (Sigma) and absorbance was examined using an ELISA reader at 450 nm.
Vaccination and HIV-1 p24 specific antibodies detection
Three females BALB/c mice, 8-week-old (purchased from the Center of Experimental Animals, Academy of Military Medical Sciences, Beijing) were injected intradermally on the back and abdomen with 80 μg purified His-tagged p24 protein mixed with complete Freund's adjuvant (100 μl per site). Pre-immune mouse sera were collected prior to immunization. After three immunizations within an interval of 21 days, these immunized mice were sacrificed. Blood samples were collected and stood at 4°C for 2 h, and the sera were aspirated after centrifugation at 4000 rpm for 10 min at 4°C. Serum samples were serially diluted from 1:500, to 1:8192000 and anti-p24 antibodies titer were determined by indirect ELISA with recombinant p24 protein. Western blotting was performed with cultured HIV-1 extracts to detect the antibodies specificity.
Construction of p24 prokaryotic expression plasmid
Expression, purification and identification of the recombinant p24 proteins
Immunoreactivity analysis of recombinant p24 protein
Efficacy of the recombinant p24 for detecting antibodies in HIV-infected individuals.
p24 ELISA result
The immunogenicity of p24 protein expressed in E.coli
The bacterial expression system is the most universally used, as it is relatively inexpensive, and with ease of manipulation and a rapid growth rate. Problematically, with the bacterial expression system, inclusion bodies are often produced due to incorrect folding and thus require denaturation and renaturation before further use. These processes may complicate or inhibit recombinant protein production. Use of a low culture temperature, low inducer concentrations and the co-expression of molecular chaperones can promote the soluble expression of recombinant proteins . In this study, the soluble p24 protein was highly expressed at a low temperature (20°C) and a low IPTG concentration (0.6 mM). To further increase the recombinant protein solubility, we chose E. coli BL21 (DE3) as the host strain, which was previously demonstrated to be appropriate for soluble expression. By adopting these strategies, the recombinant p24 protein was expressed in soluble form and was easily purified.
It is reported that protein primary structure may influence soluble protein expression. Change of the amino acids in proteins (especially cysteine) may significantly influence the folding and alter the conformation of the protein. Studies conducted by Strandberg and Wetzel et al have shown that a few amino acid changes can remarkably alter the soluble expression of target protein, and Rinas et al have also reported that individual amino acid changes involving cysteine drastically changed the solubility of recombinant proteins.
The early diagnosis of HIV infection is very important for prevention of HIV spread and ensuring safety of blood products. Currently most diagnostic reagents are based on the method of ELISA for detection of antibodies against HIV proteins[19, 23]. Synthetic peptides and/or recombinant proteins spanning the envelope (gp41 of HIV-1 and gp36 of HIV-2 respectively) and the core (p24) proteins are often used as capture antigens. Antibodies directed against p24 appear early in HIV infection and are reported to decline with progression of the disease due to increasing antigenemia. It is reported that synthetic peptides (9-53-mer) corresponding to p24 did not give satisfactory results. Recombinant p24 protein may be a better option compared with synthetic peptides. In this paper, we report the high level soluble expression of HIV-1 p24 protein in E. coli. This soluble recombinant p24 protein specifically reacts with HIV-1 infected sera. To study the immunogenicity of this soluble recombinant p24 protein, it was used to immunize mice for preparation of polyclonal antibody. Subsequent ELISA and Western-Blot analysis demonstrated that the p24 protein had proper immunogenicity in inducing mice to produce HIV-1 p24 specific antibodies. Recently the fourth generation HIV assays include the p24 antigen detection, thus high affinity antibodies with high specificity for p24 is a pre-requisite. A good recombinant protein may be a great help to manufacturing qualified p24 antibodies. Our soluble p24 protein which has exhibited good immunoreactivity and proper immunogenicity may help us to gain high affinity monoclonal antibody in the further research.
In this study, we have highly expressed soluble recombinant HIV-1 p24 protein in E.coli. This soluble p24 had good immunoreactivity and immunogenicity. Its characteristics suggest that this recombinant p24 protein holds promise for assembling the HIV diagnostic kits, as well as for the development of the fourth generation HIV test kits.
This work is supported by the Technology Major Project (2008ZX10001-013, http://www.nmp.gov.cn/), Hi-Tech Research and Development (863) Program of China (2009AA02Z111, http://program.most.gov.cn/), and National Natural Science Foundation of China (No. 30872223, http://www.nsfc.gov.cn/).
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