Pathway of Hsp70 interactions at the ribosome.

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

Expression, Rapid Purification and Functional Analysis of DnaK from Rhodococcus ruber.

BACKGROUND: Heat shock proteins (HSPs) represent a group of important proteins which are produced by all kinds of organisms especially under stressful conditions. DnaK, an Hsp70 homolog in prokaryotes, has indispensable roles when microbes was confronted with stress conditions. However, few data on DnaK from Rhodococcus sp. were available in the literature. In a previous study, we reported that toluene and phenol stress gave rise to a 29.87-fold and 3.93-fold increase for the expression of DnaK from R. ruber SD3, respectively. Thus, we deduced DnaK was in correlation with the organic solvent tolerance of R. ruber SD3. OBJECTIVE: To elucidate the role of DnaK in the organic solvent tolerance of R. ruber SD3, expression, purification and functional analysis of Dnak from R. ruber SD3 were performed in the present paper. METHODS: In this article, DnaK from R. ruber SD3 was heterologously expressed in E. coli BL21(DE3) and purified by affinity chromatography. Functional analysis of DnaK was performed using determination of kinetics, docking, assay of chaperone activity and microbial growth. RESULTS: The recombinant DnaK was rapidly purified by affinity chromatography with the purification fold of 1.9 and the recovery rate of 57.9%. Km, Vmax and Kcat for Dnak from R. ruber SD3 were 80.8 µM, 58.1 nmol/min and 374.3 S-1, respectively. The recombinant protein formed trimer in vitro, with the calculated molecular weight of 214 kDa. According to in-silico analysis, DnaK interacted with other molecular chaperones and some important proteins in the metabolism. The specific activity of catalase in the presence of recombinant DnaK was 1.85 times or 2.00 times that in the presence of BSA or Tris-HCl buffer after exposure to 54 °C for 1h. E. coli transformant with pET28-dnak showed higher growth than E. coli transformant with pET28 at 43°C and in the presence of phenol, respectively. CONCLUSION: The biochemical properties and the interaction analysis of DnaK from R. ruber SD3 deepened our understanding of DnaK function. DnaK played an important role in microbial growth when R. ruber was subjected to various stress such as heating and organic solvent.

The Role of p53 Protein in the Realization of the Exogenous Heat Shock Protein 70 Anti-Apoptotic Effect during Axotomy.

The search for effective neuroprotective agents for the treatment of neurotrauma has always been of great interest to researchers around the world. Extracellular heat shock protein 70 (eHsp70) is considered a promising agent to study, as it has been demonstrated to exert a significant neuroprotective activity against various neurodegenerative diseases. We showed that eHsp70 can penetrate neurons and glial cells when added to the incubation medium, and can accumulate in the nuclei of neurons and satellite glial cells after axotomy. eHsp70 reduces apoptosis and necrosis of the glial cells, but not the neurons. At the same time, co-localization of eHsp70 with p53 protein, one of the key regulators of apoptosis, was noted. eHsp70 reduces the level of the p53 protein apoptosis promoter both in glial cells and in the nuclei and cytoplasm of neurons, which indicates its neuroprotective effect. The ability of eHsp70 to reverse the proapoptotic effect of the p53 activator WR1065 may indicate its ability to regulate p53 activity or its proteosome-dependent degradation.

A novel method for removing contaminant Hsp70 molecular chaperones from recombinant proteins.

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.

Genotypic Characterization of Cryptosporidium Species in Humans and Peri-Domestic Animals in Ekiti and Oyo States, Nigeria.

Cryptosporidiosis is one of the leading causes of diarrhea in humans and several other vertebrate species. Because surveys of Cryptosporidium genotypes from animals and humans living in the same region are rare, our understanding of the importance of zoonotic transmission in the epidemiology of cryptosporidiosis remains superficial. PCR was used to amplify a portion of the Cryptosporidium 18S small subunit ribosomal RNA gene from fecal DNA from humans and livestock living in Ekiti and Oyo states, Nigeria. PCR-positive samples were further analyzed using PCR targeting the heat-shock protein HSP-70, the actin, and the sporozoite glycoprotein gene gp60. A questionnaire was used to collect demographic information. Sixteen of 187 samples collected were Cryptosporidium 18S PCR positive. Of these, 5 samples originating from HIV-positive patients, 5 from otherwise healthy children, 2 from chickens, 3 from goats, and 1 from a dog were positive for at least 1 marker. Sequencing of the 18S rRNA amplicons revealed the presence of Cryptosporidium parvum in 2 HIV positive patients and in a child; the actin sequence confirmed the presence of this species. Two samples of HIV-positive patients amplified Cryptosporidium hominis 18S rRNA, one of them confirmed by the HSP-70, actin, and gp60 sequences. Cryptosporidium meleagridis was found in another HIV patient, while C. hominis was detected in 3 children (of which 2 were confirmed by gp60). Cryptosporidium muris was found in 1 child. In birds, we found C. meleagridis and, significantly, C. parvum, whereas we detected C. parvum and C. muris in 1 goat each. The only dog sampled was positive for Cryptosporidium canis. We conclude that, in the environment we surveyed, humans and animals are a potential part of the same transmission cycle. Measures to prevent zoonotic transmission should therefore be considered to reduce the prevalence of cryptosporidiosis.

A highly efficient, one-step purification of the Hsp70 chaperone Ssa1.

Chaperone proteins are required to maintain the overall fold and function of proteins in the cell. As part of the Hsp70 family, Ssa1 acts to maintain cellular proteostasis through a variety of diverse pathways aimed to preserve the native conformation of target proteins, thereby preventing aggregation and future states of cellular toxicity. Studying the structural dynamics of Ssa1 in vitro is essential to determining their precise mechanisms and requires the development of purification methods that result in highly pure chaperones. Current methods of expressing and purifying Ssa1 utilize affinity tagged constructs expressed in Escherichia coli, however, expression in an exogenous source produces proteins that lack post-translational modifications leading to undesired structural and functional effects. Current protocols to purify Ssa1 from Saccharomyces cerevisiae require large amounts of starting material, multiple steps of chromatography, and result in low yield. Our objective was to establish a small-scale purification of Ssa1 expressed from its endogenous source, Saccharomyces cerevisiae, with significant yield and purity. We utilized a protein A affinity tag that was previously used to purify large protein complexes from yeast, combined with magnetic Dynabeads that are conjugated with rabbit immunoglobulin G (IgG). Our results show that we can produce native, highly pure, active Ssa1 via this one-step purification with minimal amounts of starting material, and this Ssa1-protein A fusion does not alter cellular phenotypes. This methodology is a significant improvement in Ssa1 purification and will facilitate future experiments that will elucidate the biochemical and biophysical properties of Hsp70 chaperones.

HSP70 Copurifies with Zika Virus Particles.

Zika virus (ZIKV) has been identified as a cause of neurologic diseases in infants and Guillain-Barré Syndrome, and currently, no therapeutics or vaccines are approved. In this study, we sought to identify potential host proteins interacting with ZIKV particles to gain better insights into viral infectivity. Viral particles were purified through density-gradient centrifugation and subsequently, size-exclusion chromatography (SEC). Mass spectrometric analyses revealed viral envelope protein and HSP70 to comigrate in only one SEC fraction. Neither of these proteins were found in any other SEC fractions. We then performed neutralization assays and found that incubating viral particles with antibody against HSP70 indeed significantly reduced viral infectivity, while HSC70 antibody did not. Preincubating cells with recombinant HSP70 also decreased viral infectivity. Knockdown and inhibition of HSP70 also significantly diminished viral production. These results implicate HSP70 in the pathogenesis of ZIKV and identify HSP70 as a potential host therapeutic target against ZIKV infection.

Molecular cloning, prokaryotic expression, purification, structural studies and functional implications of Heat Shock Protein 70 (Hsp70) from Rutilus frisii kutum.

A novel Hsp70 chaperone from Rutilus frisii kutum was identified, cloned, expressed, purified and its functional characteristics revealed. The 3D structure of Hsp70 from Rutilus kutum was constructed using the crystal structure of E. coli Hsp70 as the template, with 47% sequence identity. The in vitro ATPase activity assay after 60min, ATP hydrolysis of purified recombinant Hsp70 (8µM) was improved by binding to denatured thermally luciferase (3µM) about 2.5-fold compared with that of Hsp70 alone. Based on the results, it was found that the purified Hsp70 chaperone was able to considerably suppress heat-induced aggregation of luciferase by binding to DnaJ co-chaperone (5µM) more than 70% after 10min at 42°C. In addition, Hsp70 DnaJ complex improved the refolding of heat-shocked luciferase nearly 40% after 60min at 25°C. It was concluded that Hsp70 protein from Rutilus frisii kutum has the critical role in preventing heat-induced aggregation of luciferase and refolding of heat-denatured luciferase was strictly dependent on the activity of Hsp70, thus, this protein can potentially be used for improving the functional properties of luciferase in various applications.

Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis.

Human mortalin is a Hsp70 mitochondrial protein that plays an essential role in the biogenesis of mitochondria. The deregulation of mortalin expression and its functions could lead to several age-associated disorders and some types of cancers. In the present study, we optimized the expression and purification of recombinant human mortalin by the use of two-step chromatography. Low temperature (18°C) and 0.5mM (IPTG) was required for optimum mortalin expression. Chaperone activity of mortalin was assessed by the citrate synthase and insulin protection assay, which suggested their protective role in mitochondria. Folding and unfolding assessments of mortalin were carried out in the presence of guanidine hydrochloride (GdnHCl) by intrinsic fluorescence measurement, ANS (8-analino 1-nephthlene sulfonic acid) binding and CD (circular dichroism) analysis. Under denaturing conditions, mortalin showed decrease in tryptophan fluorescence intensity along with a red shift of 11nm. Moreover, ANS binding studies illustrated decrease in hydrophobicity. CD measurement of mortalin showed a predominant helical structure. However, the secondary structure was lost at low concentration of GdnHCl (1M). We present a simple and robust method to produce soluble mortalin and warranted that chaperones are also susceptible to unfolding and futile to maintain protein homeostasis.

Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene.

Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.

[Preparation of chaperone-antigen peptide vaccine derived from human gastric cancer stem cells and its immune function].

Objective: To explore the method of extracting chaperone antigen peptide complexes from gastric cancer stem cells and its immune function. Methods: Gastric cancer stem cells and gastric cancer cells were screened by low temperature ultrasonic lysis. After salting out and dialysis, the lysate supernatant was processed with SDS-PAGE to analyze the expression of chaperone antigen peptide complexes, and then was separated and purified with CNBr-activated SepharoseTM 4B. Reverse high pressure liquid chromatography (HPLC), SDS-PAGE and Western blotting were used to analyze the purity and nature of the acquired albumen. Lymphocyte proliferation assay and lymphocytotoxicity assay were used to ditermine the immunological activity of the chaperone-antigen peptide complexes. Results: The chaperone antigen peptide complexes of gastric cancer stem cells were prepared and identified successfully, of which the main components were the antigen peptides of HSP60, HSP70, HSP90 and HSP110. 0.75 µg and 1.00 µg HSP70-antigen peptide and 1.00 µg HSP90-antigen peptide activated lymphocytes significantly. Their A(490) values were 0.26±0.03, 0.45±0.05 and 0.32±0.04, respectively, while the corresponding doses of HSP60-antigen peptide and HSP110-antigen peptide did not activate lymphocytes. The killing rates of 1.00 µg HSP70-antigen peptide and 1.00 µg HSP70 were (45.0±2.0)% and (16.0±2.0)%, respectively, showing a significant difference (P=0.012). Similarly, the killing rates of 1.00 µg HSP90-antigen peptide and 1.00 µg HSP90 were (36.0±5.0)% and (13.0±4.0)%, respectively, also showing a significant difference (P=0.048). Conclusions: The amount of chaperone antigen peptide complexes in gastric cancer cells is extremely low, but it is obviously increased in gastric cancer stem cells. After purification, the chaperone antigen peptide complexes with high purity can be prepared. The extracted chaperone antigen peptide complexes have stronger immunogenicity, and can be used to make tumor vaccine in vitro, which may have a good application value in the targeted therapy of gastric cancer.

Recombinant human Tat-Hsp70-2: A tool for neuroprotection.

Human Hsp70-2 is a chaperone expressed mainly in the nervous system. Up to now, no study has reported on the recombinant expression of this important human chaperone. Herein, we describe the successful purification and characterization of recombinant human Hsp70-2 in Escherichia coli in both the full-length and the chimeric protein containing the protein transduction domain corresponding to the trans-activator of transcription (Tat) from HIV. Under optimized conditions, the Tat-Hsp70-2 was expressed in a soluble form and purified by two chromatographic steps (in a 3.6 mg/L fermentation broth yield): recombinant Tat-Hsp70-2 was folded and showed ATPase activity. In contrast, the full-length recombinant protein was only expressed in the form of inclusion bodies and thus was purified following a refolding procedure. The refolded Hsp70-2 protein was inactive and the protein conformation slightly altered as compared to the corresponding Tat-fused variant. The Tat-Hsp70-2 protein (100 nM), when added to human neuroblastoma SH-SY5Y cells subjected to hydrogen peroxide or 6-hydroxydopamine stress, partially protected from the deleterious effect of these treatments. This work describes an approach for the functional expression of human Tat-Hsp70-2 that provides sufficient material for detailed structure-function studies and for testing its ability to protect neuroblastoma cells from oxidative stress.

A Proteomic-Based Workflow Using Purified Respiratory Syncytial Virus Particles to Identify Cellular Factors as Drug Targets.

The identification of cellular factors that play a role in respiratory syncytial virus (RSV) replication is an alternative strategy in the identification of druggable cellular protein that are essential for RSV replication. In this regard experimental strategies that are able to screen relevant proteins from the vast array of proteins in the cellular milieu will facilitate the identification of potential drug targets. In this chapter we describe a procedure where RSV particles are purified from cells that are permissive for RSV infection, and the protein composition of the purified virus particles characterized using a proteomics-based strategy. This procedure revealed that actin, several actin-binding proteins, and the chaperones HSP70 and HSP90 also co-purified with the virus particles. The relevance of the HSP90 protein to virus replication was then further validated using imaging, gene silencing and by using an established small molecule HSP90 inhibitor.

The Malarial Exported PFA0660w Is an Hsp40 Co-Chaperone of PfHsp70-x.

Plasmodium falciparum, the human pathogen responsible for the most dangerous malaria infection, survives and develops in mature erythrocytes through the export of proteins needed for remodelling of the host cell. Molecular chaperones of the heat shock protein (Hsp) family are prominent members of the exportome, including a number of Hsp40s and a Hsp70. PFA0660w, a type II Hsp40, has been shown to be exported and possibly form a complex with PfHsp70-x in the infected erythrocyte cytosol. However, the chaperone properties of PFA0660w and its interaction with human and parasite Hsp70s are yet to be investigated. Recombinant PFA0660w was found to exist as a monomer in solution, and was able to significantly stimulate the ATPase activity of PfHsp70-x but not that of a second plasmodial Hsp70 (PfHsp70-1) or a human Hsp70 (HSPA1A), indicating a potential specific functional partnership with PfHsp70-x. Protein binding studies in the presence and absence of ATP suggested that the interaction of PFA0660w with PfHsp70-x most likely represented a co-chaperone/chaperone interaction. Also, PFA0660w alone produced a concentration-dependent suppression of rhodanese aggregation, demonstrating its chaperone properties. Overall, we have provided the first biochemical evidence for the possible role of PFA0660w as a chaperone and as co-chaperone of PfHsp70-x. We propose that these chaperones boost the chaperone power of the infected erythrocyte, enabling successful protein trafficking and folding, and thereby making a fundamental contribution to the pathology of malaria.

Eukaryotic Hsp70 chaperones in the intermembrane space of chloroplasts.

MAIN CONCLUSION: Multiple eukaryotic Hsp70 typically localized in the cytoplasm are also distributed to the intermembrane space of chloroplasts and might thereby represent the missing link in energizing protein translocation. Protein translocation into organelles is a central cellular process that is tightly regulated. It depends on signals within the preprotein and on molecular machines catalyzing the process. Molecular chaperones participate in transport and translocation of preproteins into organelles to control folding and to provide energy for the individual steps. While most of the processes are explored and the components are identified, the transfer of preproteins into and across the intermembrane space of chloroplasts is not yet understood. The existence of an energy source in this compartment is discussed, because the required transit peptide length for successful translocation into chloroplasts is shorter than that found for mitochondria where energy is provided exclusively by matrix chaperones. Furthermore, a cytosolic-type Hsp70 homologue was proposed as component of the chloroplast translocon in the intermembrane space energizing the initial translocation. The molecular identity of such intermembrane space localized Hsp70 remained unknown, which led to a controversy concerning its existence. We identified multiple cytosolic Hsp70s by mass spectrometry on isolated, thermolysin-treated Medicago sativa chloroplasts. The localization of these Hsp70s of M. sativa or Arabidopsis thaliana in the intermembrane space was confirmed by a self-assembly GFP-based in vivo system. The localization of cytosolic Hsp70s in the stroma of chloroplasts or different mitochondrial compartments could not be observed. Similarly, we could not identify any cytosolic Hsp90 in the intermembrane space of chloroplast. With respect to our results we discuss the possible targeting and function of the Hsp70 found in the intermembrane space.

Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells.

The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

Production and preliminary evaluation of Trypanosoma evansi HSP70 for antibody detection in Equids.

The present immuno-diagnostic method using soluble antigens from whole cell lysate antigen for trypanosomosis have certain inherent problems like lack of standardized and reproducible antigens, as well as ethical issues due to in vivo production, that could be alleviated by in vitro production. In the present study we have identified heat shock protein 70 (HSP70) from T. evansi proteome. The nucleotide sequence of T. evansi HSP70 was 2116 bp, which encodes 690 amino acid residues. The phylogenetic analysis of T. evansi HSP70 showed that T. evansi occurred within Trypanosoma clade and is most closely related to T. brucei brucei and T. brucei gambiense, whereas T. congolense HSP70 laid in separate clade. The two partial HSP70 sequences (HSP-1 from N-terminal region and HSP-2 from C-terminal region) were expressed and evaluated as diagnostic antigens using experimentally infected equine serum samples. Both recombinant proteins detected antibody in immunoblot using serum samples from experimental infected donkeys with T. evansi. Recombinant HSP-2 showed comparable antibody response to Whole cell lysate (WCL) antigen in immunoblot and ELISA. The initial results indicated that HSP70 has potential to detect the T. evansi infection and needs further validation on large set of equine serum samples.

Prophylactic Admission of an In Vitro Reconstructed Complexes of Human Recombinant Heat Shock Proteins and Melanoma Antigenic Peptides Activates Anti-Melanoma Responses in Mice.

Tumor-derived autologous antigenic peptides when bound to endogenous 70 kDa family heat shock proteins (HSP70) are able to induce effective T-cell responses against tumors. However, efficacy of HSPbased vaccines in clinical practical stand point still has a number of certain limitations including an activation of immune responses against alien non-human HSPs. In this study we reconstructed the complexes of human recombinant HSPs70 (human recombinant HSP70A1B and HSC70 mixture; hrHSPs70) with antigenic lowweight peptides derived from mice B16F10 melanoma cell lysate (PepMCL) in vitro and investigated the prophylactic potential of these complexes to activate anti-tumor immunity in melanoma mouse model. Our results demonstrate that the developed prophylactic vaccine elicits melanoma-specific immune responses and anti-tumor effects against melanoma. These results suggest that hrHSPs70 has capability to reconstitute complexes with peptides obtained from tumor cells lysates in vitro and, therefore, can be used for delivery of multiple antigenic peptides into antigen-presenting cells (APCs) to activate effectors cells. Designed in such a way hrHSPs70-based prophylactic vaccines induce immune responses resulting in a significant efficient prevention of tumor growth and metastases.

Overexpression, Purification and Characterisation of the Plasmodium falciparum Hsp70-z (PfHsp70-z) Protein.

Six Hsp70-like genes are represented on the genome of Plasmodium falciparum. Of these two occur in the cytosol: P. falciparum Hsp70-z (PfHsp70-z) and PfHsp70-1. PfHsp70-1 is a well characterised canonical Hsp70 that facilitates protein quality control and is crucial for the development of malaria parasites. There is very little known about PfHsp70-z. However, PfHsp70-z is known to be essential and is implicated in suppressing aggregation of asparagine-rich proteins of P. falciparum. In addition, its expression at the clinical stage of malaria correlates with disease prognosis. Based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins. Since Hsp110 proteins have been described as nucleotide exchange factors (NEFs) of their canonical Hsp70 counterparts, it has been speculated that PfHsp70-z may serve as a NEF of PfHsp70-1. In the current study, P. falciparum cells cultured in vitro were subjected to heat stress, triggering the enhanced expression of PfHsp70-z. Biochemical assays conducted using recombinant PfHsp70-z protein demonstrated that the protein is heat stable and possesses ATPase activity. Furthermore, we observed that PfHsp70-z is capable of self-association. The structural-functional features of PfHsp70-z provide further evidence for its role as a chaperone and possible nucleotide exchange factor of PfHsp70-1.

Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot.

This paper describes the application of an automated size-based capillary Western blot system (Sally instrument) from ProteinSimple, Inc., for biopharmaceutical fusion-Fc protein characterization and evaluation of its purification process. The fusion-Fc protein column purification from an excess of single chain Fc polypeptide and removal of an enzyme coexpressed for protein maturation have been demonstrated using an automated capillary Western system. The clearance of a selected host cell protein (HCP) present in cell culture of fusion-Fc protein was also quantitatively monitored throughout the protein purification process. Additionally, the low levels of fusion-Fc product-related impurities detected by traditional slab gel Western blot were confirmed by the automated capillary Western system. Compared to the manual approach, the automated capillary Western blot provides the advantages of ease of operation, higher sample throughput, greater linearity range, and higher precision for protein quantitation.