Recombinant P24 Research Articles

Protein binding protection in combination with DNA masking for sensitive and reliable transcription factor detection

Sensitive and reliable detection of transcription factors (TFs) is crucial for disease diagnosis and drug discovery. Herein, a protein binding protection in combination with DNA masking-based strategy is developed for sensitive and reliable detection of NF-κB p50. First, NF-κB p50 binds specifically to a hairpin probe to form a protein-DNA complex. Then, the formed complex protects the hairpin probe from hybridization with the masking strand and subsequently triggers the cascade signal amplification of strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Excess hairpin probes that do not bind NF-κB p50 are masked through hybridization with masking strands into stable duplexes, prohibiting the non-specific amplification and avoiding the risk of false positive signals. The signal results from the presence of NF-κB p50, ensuring the detection reliability. Through the cascade amplification specifically triggered by the target, the method can detect the purified recombinant NF-κB p50 down to 1.0 × 10–13 M. It is further employed for the NF-κB p50 inhibitor screening and analysis, which shows the potential in anti-NF-κB p50 drug discovery. Moreover, the method is applied in detection NF-κB p50 in cell nuclear extracts with a detection limit of 0.1 ng μL−1. The proposed strategy will provide a promising tool for sensitive and reliable assaying NF-κB p50 activity in biomedical study and disease diagnosis.

RSjP40 protein promotes PPARγ expression in LX-2 cells through microRNA-27b.

miR-27b is reported to participate in the proliferation and differentiation of hepatic stellate cells (HSCs) and to regulate fat metabolism of rat HSCs by targeting retinoid X receptor α. Our previous study also indicated that the recombinant P40 protein from Schistosoma japonicum (rSjP40) inhibited the activation of HSCs. In this study, we observed the expression of miR-27b in rSjP40-treated LX-2 cells and explored its potential mechanisms. Quantitative real-time PCR showed that rSjP40 inhibits the expression of miR-27b in LX-2 cells. Further results obtained by Western blot and dual-luciferase reporter assay confirmed that miR-27b regulates peroxisome proliferator-activated receptor γ (PPARγ) expression in rSjP40-treated LX-2 cells by targeting the 3'-UTR of PPARγ. 5-AZA-2'-deoxycytidine (5-AZA-dC), which inhibits methylation of HSCs, partially reversed rSjP40-induced down-regulation expression of miR-27b in LX-2 cells. 5-AZA-dC also partially reversed rSjP40-induced up-regulation expression of PPARγ in LX-2 cells. The increased expression of PPARγ in rSjP40-treated LX-2 cells may be partially due to miR-27b methylation. Therefore, our study provides further insight into the mechanism by which rSjP40 inhibits HSC activation and provides a basis for future study of the blocking effect of rSjP40 in liver fibrosis.-Zhu, D., Lyu, L., Shen, P., Wang, J., Chen, J., Sun, X., Chen, L., Zhang, L., Zhou, Q., Duan, Y. rSjP40 protein promotes PPARγ expression in LX-2 cells through microRNA-27b.

Interaction of the middle domains stabilizes Hsp90α dimer in a closed conformation with high affinity for p23.

The human genome encodes two highly similar cytosolic Hsp90 proteins called isoforms Hsp90α and Hsp90β. Of the 300 client proteins for Hsp90 identified so far only a handful interact specifically with one Hsp90 isoform. Here we report for the first time that Hsp90 cochaperone p23 binds preferentially to Hsp90α and that this interaction is mediated by the middle domain of Hsp90α. Based on the homology modeling, we infer that the middle domains in the Hsp90α dimer bind stronger with each other than in the Hsp90β dimer. Therefore, compared to Hsp90β, Hsp90α may adopt closed conformation more easily. Hsp90 interacts with p23 in the closed conformation. Hsp90α binds human recombinant p23 about three times stronger than Hsp90β but with significantly smaller exothermic enthalpy as determined by isothermal titration calorimetry of direct binding between the purified proteins. As p23 binds to Hsp90 in a closed conformation, stabilization of the Hsp90α dimer in the closed conformation by its middle domains explains preference of p23 to this Hsp90 isoform.

Expression of Human Immunodeficiency Virus type 1 (HIV-1) coat protein genes in plants using cotton leaf curl Multan betasatellite-based vector.

It has already been demonstrated that a betasatellite associated with cotton leaf curl Multan virus (CLCuMB) can be used as a plant and animal gene delivery vector to plants. To examine the ability of CLCuMB as a tool to transfer coat protein genes of HIV-1 to plants, two recombinant CLCuMB constructs in which the CLCuMB βC1 ORF was replaced with two HIV-1 genes fractions including a 696 bp DNA fragment related to the HIV-1 p24 gene and a 1501 bp DNA fragment related to the HIV-1 gag gene were constructed. Gag is the HIV-1 coat protein gene and p24 is a component of the particle capsid. Gag and p24 are used for vaccine production. Recombinant constructs were inoculated to Nicotiana glutinosa and N. benthamiana plants in the presence of an Iranian isolate of Tomato yellow leaf curl virus (TYLCV-[Ab]) as a helper virus. PCR analysis of inoculated plants indicated that p24 gene was successfully replicated in inoculated plants, but the gag gene was not. Real-time PCR and ELISA analysis of N. glutinosa and N. benthamiana plants containing the replicative forms of recombinant construct of CLCuMB/p24 indicated that p24 was expressed in these plants. This CLCuMB-based expression system offers the possibility of mass production of recombinant HIV-1 p24 protein in plants.

Comparative studies of the low-resolution structure of two p23 co-chaperones for Hsp90 identified in Plasmodium falciparum genome

The p23 proteins are small acidic proteins that aid the functional cycle of the Hsp90 molecular chaperone. Such co-chaperone acts by temporarily inhibiting the ATPase activity of Hsp90 and exhibits intrinsic chaperone activity, suggesting independent roles. A search for p23 in the Plasmodium falciparum genome led to the identification of two putative proteins showing 13% identity to each other and approximately 20% identity to human p23. To understand the presence of two p23 proteins in this organism, we generated recombinant p23 proteins (Pfp23A and Pfp23B) and investigated their structure and function. The proteins presented some similarities and dissimilarities in structural contents and showed different chemical and thermal stabilities, with Pfp23A being more stable than Pfp23B, suggesting that these proteins may present different functions in this organism. Both Pfp23 proteins behaved as elongated monomers in solution and were capable of preventing the thermal-induced aggregation of model client proteins with different efficiencies. Finally, the Pfp23 proteins inhibited the ATPase activity of recombinant P. falciparum Hsp90 (PfHsp90). These results validate the studied proteins as p23 proteins and co-chaperones of PfHsp90.

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats

The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.

Expression of Functional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells.

Purpose: More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1. Methods: The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells. Results: Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%. Conclusion: Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1.

Recombinant SjP40 protein enhances p27 promoter expression in hepatic stellate cells via an E2F1-dependent mechanism.

The p27 protein plays a critical role in cell cycle arrest. Our previous studies have demonstrated that recombinant P40 protein from Schistosoma japonicum (rSjP40) could induce G1 phase arrest of cell cycle. We, therefore, attempted to observe the effect of rSjP40 on p27 promoter activity in LX-2 cells and to explore its potential mechanisms in this study. Using both Western blot and dual-luciferase reporter assay, we demonstrated that rSjP40 could enhance the expression of p27 in LX-2 cells. Results obtained using truncated fragments of p27 promoter showed that rSjP40 increased p27 promoter activity in LX-2 cells, mainly via some transcription factors that bind to the -1740/-873 region of p27 promoter. Further studies confirmed that the enhancement of p27 promoter activity induced by rSjP40 was related to E2F1 in LX-2 cells. Transfection of siRNA of E2F1 could also restore the effect of rSjP40 on expression of p27 and partially on α-SMA. Therefore, our study provided further insights into the mechanism by which rSjP40 induces LX-2 cell cycle arrest at G1 phase and inhibits HSC activation. Our results provide basis for future study of the blocking effect of rSjP40 in liver fibrosis.

AN EVALUATION OF ELISA USING RECOMBINANT P17 ANTIGEN FOR CATTLE BRUCELLOSIS

Brucellae are Gram-negative cocccobacilli, facultative intracellular bacterial pathogens of both humans and animals. Brucellosis is an important disease that is difficult to diagnose and treat that causes heavy economic losses and human suffering. Diagnosis of brucellosis plays a vital role for control and prevention of the disease. Lipopolysaccharide (LPS) based Enzyme Linked Immuno Sorbent Assay (ELISA) shows false positivity due to cross-reactivity with other gram-negative bacteria LPS. The present investigation was undertaken to assess the diagnostic potential of the recombinant P17 protein of Brucella. P17 gene of Brucella abortus (B. abortus) was amplified, cloned and subcloned into pQE 30 vector yielding high levels of protein expression. The purified recombinant P17 (rP17) protein was used to develop an indirect ELISA (i-ELISA) test for brucellosis. The rP17-ELISA was compared with RBPT (Rose Bengal Precipitation Test) and LPS-ELISA using 530 cattle sera. The concordance percentage and kappa statistics of P17-ELISA is greater in compression with LPS-ELISA. Relative sensitivity and relative specificity of P17-ELISA shows a positive trend with RBPT. The data suggest that P17-ELISA can be a useful method for Brucella diagnosis and recombinant P17 protein is a potential antigen for diagnosis of cattle brucellosis.

Novel Therapeutic Approach for Inhibition of HIV-1 Using Cell-Penetrating Peptide and Bacterial Toxins.

Despite advancements in our understanding of HIV-1 pathogenesis, critical virus components for immunity, vaccines trials, and drugs development, challenges remain in the fight against HIV-1. Of great importance is the inhibitory function of microbicidal cell penetrating peptides and bacterial toxins that interfere with production and neutralize infection of HIV-1 particles. We demonstrate that the neutralizing activity of a cationic 18 amino acids peptide, is similar to a broadly neutralizing human antibody, and inhibits production of two HIV-1 strains in human cell lines. Pretreatment of cells with bacterial toxins or toxoids derived from enterotoxigenic E. coli, boost subsequent activity of the peptide against HIV-1, to inhibit simultaneously production and infection. The synthetic peptide crosses the cell membrane into the cytoplasm and nucleus. In vitro analysis of a possible target for this peptide revealed specific binding to recombinant HIV-1 gag p24. This is the first demonstration of a synergy between bacterial toxins and a cell-penetrating peptide against HIV-1.

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China.

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560–616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.

Recombinant Protein p30 for Serological Diagnosis of African Swine Fever by Immunoblotting Assay.

This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly-histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30-IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30-IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30-IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30-IB allowed the detection of ASFV-specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8days post-infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south-east Africa).

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization

ABSTRACTContagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG’s and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at ∼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum.

Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70days to parturition every 2weeks. After parturition, each calf received his dam colostrum and challenged with 1×10(7) Cryptosporidium parvum oocysts at 12h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum.

Validation according to OIE criteria of a monoclonal, recombinant p26–based, serologic competitive enzyme-linked immunosorbent assay as screening method in surveillance programs for the detection of Equine infectious anemia virus antibodies

The Italian National Reference Center for equine infectious anemia (CRAIE; Rome, Italy) developed and validated a monoclonal, recombinant p26-based competitive enzyme-linked immunosorbent assay (cELISA) for the detection of EIA virus antibodies employing the 2010 criteria of the World Organization for Animal Health (OIE). The following parameters were evaluated: cutoff values, repeatability, reproducibility, concordance, analytical sensitivity (Se), absolute analytical specificity (Sp), and diagnostic Se and Sp. Positive and negative predictive values were also defined in relation to the estimated prevalence. When the cELISA was used as a screening test for 96,468 samples in the Italian EIA surveillance program, 17% more EIA cases were detected than by the agar gel immunodiffusion test, and the apparent diagnostic Sp estimated from these samples was 99.8%, which was more than the diagnostic Sp (80.2%) estimated from validation. The high Se and Sp of the cELISA confirm its fit for purpose as a screening test.

New p32/gC1qR Ligands for Targeted Tumor Drug Delivery.

Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.

Molecular Validation of Putative Antimicrobial Peptides for Improved Human Immunodeficiency Virus Diagnostics via HIV Protein p24

Objective: Various HIV diagnostic systems are available however; the p24 antigen detection assay is preferred due to its ability to decrease the window period for HIV detection. The p24 antigen assay has been associated with low sensitivity. Antimicrobial Peptides (AMPs) display huge potential in development of more effective diagnostics tools. The aim of this study was to design derivative AMPs from AMPs identified previously, using an in silico approach, as templates, with higher predicted binding affinity for the p24 protein and implement the best suited AMPs in a Point-Of- Care (POC) device for detection of HIV-1 and HIV-2. Method: This study firstly used an in silico approach to identify derivative AMPs which bind the N-terminal domain of the p24 antigen with increased predicted binding affinity. The in silico approach used the parental AMPs as templates for generation of the derivative AMPs through site-directed mutagenesis, 3-D structure prediction and docking studies. Secondly, the binding between the synthetically synthesized AMPs and the HIV antigen p24 was validated by an in house Lateral Flow Device (LFD) “on/off” binding experiment. The most promising AMPs were conjugated to gold nanoparticles (AuNPs) and implemented in a LFD to detect HIV-1 and HIV-2 using patient sera as well as a panel of international HIV standards (panel C10). Result: With conjugation of AMPs to gold nanoparticles, parent AMP 1 and derivative AMP 1.1 used in combination most sensitively detected the recombinant protein p24 and was therefore selected for development of a LFD prototype for HIV diagnostics. Global HIV-1 (C10) and HIV-2 (C10) standards were successfully detected by the LFD prototype as well as HIV infected serum samples. Conclusion: A sensitive HIV-1 and HIV-2 LFD prototype was developed which would within 15 minutes provide patients with accurate and sensitive diagnosis of HIV.

A New Controversial Pathophysiology Confirms that HIV does not Kill the CD4+ T-Cell But Mutates Its Physiological Behavior Becoming an Unaccountable CD8+ T-Cell

HIV pathogenesis is known to cause a progressive depilation of CD4+ T-cell cell population in close association with progressive impairment of cellular immunity and increase the susceptibility to infection. This new study is giving a new explanation about the mode of action of this virus. We assume that there is an alteration in the physiological behavior of CD4+ T-cells causing it to mutate to CD8+ T-cells and that CD4+ T-cell are neither destroyed nor lost during the infection. Twelve consenting adults took part in a randomized control trail, six were tested positive for HIV and had never received any antiretroviral therapy while the other six were tested negative. Blood samples withdrawn from participants were tested for total CD4+ T-cells and CD8+ T-cells .Infected cells from HIV positive patients were stimulated with a purified recombinant HIV-1 p17 matrix as a viral protein along with other immunological assays. The collected data showed that the sum of both CD4+ T-cells and CD8+ T-cells did not change in HIV positive patients, although there were a decrease in CD4+ T-cells and an increase in CD8+ T-cell count. Our study confirms that CD8+ T-cells is responsible for the increase in scope of HIV and the susceptibility to (OI), we assumed that this resulted from the duplication in cell signals of both newly formed (mutant) and originally found CD8+ T-cells causing a complete cellular discrepancy. According to our findings a new area of medications could arise to be a promising therapeutical modality for treating HIV-1 infection.

Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities.

The small acidic protein called p23 acts as a co-chaperone for heat-shock protein of 90 kDa (Hsp90) during its ATPase cycle. p23 proteins inhibit Hsp90 ATPase activity and show intrinsic chaperone activity. A search for p23 in protozoa, especially trypanosomatids, led us to identify two putative proteins in the Leishmania braziliensis genome that share approximately 30% identity with each other and with the human p23. To understand the presence of two p23 isoforms in trypanosomatids, we obtained the recombinant p23 proteins of L. braziliensis (named Lbp23A and Lbp23B) and performed structural and functional studies. The recombinant proteins share similar solution structures; however, temperature- and chemical-induced unfolding experiments showed that Lbp23A is more stable than Lbp23B, suggesting that they may have different functions. Lbp23B prevented the temperature-induced aggregation of malic dehydrogenase more efficiently than did Lbp23A, whereas the two proteins had equivalent efficiencies with respect to preventing the temperature-induced aggregation of luciferase. Both proteins interacted with L. braziliensis Hsp90 (LbHsp90) and inhibited its ATPase activity, although their efficiencies differed. In vivo identification studies suggested that both proteins are present in L. braziliensis cells grown under different conditions, although Lbp23B may undergo post-translation modifications. Interaction studies indicated that both Lbp23 proteins interact with LbHsp90. Taken together, our data suggest that the two protozoa p23 isoforms act similarly when regulating Hsp90 function. However, they also have some differences, indicating that the L. braziliensis Hsp90 machine has features providing an opportunity for novel forms of selective inhibition of protozoan Hsp90.