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.

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.

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.

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.

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.

Overproduction and biophysical characterization of human HSP70 proteins.

Heat shock proteins (HSP) perform vital cellular functions and modulate cell response pathways to physical and chemical stressors. A key feature of HSP function is the ability to interact with a broad array of protein binding partners as a means to potentiate downstream response pathways or facilitate protein folding. These binding interactions are driven by ATP-dependent conformational rearrangements in HSP proteins. The HSP70 family is evolutionarily conserved and is associated with diabetes and cancer progression and the etiopathogenesis of hepatic, cardiovascular, and neurological disorders in humans. However, functional characterization of human HSP70s has been stymied by difficulties in obtaining large quantities of purified protein. Studies of purified human HSP70 proteins are essential for downstream investigations of protein-protein interactions and in the rational design of novel family-specific therapeutics. Within this work, we present optimized protocols for the heterologous overexpression and purification of either the nucleotide binding domain (NBD) or the nucleotide and substrate binding domains of human HSPA9, HSPA8, and HSPA5 in either Escherichia coli or Saccharomyces cerevisiae. We also include initial biophysical characterization of HSPA9 and HSPA8. This work provides the basis for future biochemical studies of human HSP70 protein function and structure.

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.

Isolation and amplification of mRNA within a simple microfluidic lab on a chip.

The major modules for realizing molecular biological assays in a micro-total analysis system (µTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.

Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli.

[Stress proteins in the cells of Porphyra purpurea (Rhodophyta) thallus].

Heat shock proteins have been revealed for the first time by the methods of Western blotting using alkaline phosphatase and ECL in the cells of Porphyra purpurea from Kattegat area of the Baltic Sea in normal and experimental stress conditions. It was demonstrated with application of monoclonal anti-Hsp70 antibodies that a slight band about 70 kDa is present constitutively at the film; additionally the polypeptide of about 40 kDa ("Hsp40") has been detected. After heat shock at 28 degrees C during 1 hr significant "expenditure" of Hsp70 was observed, as well as the pronounced induction of "Hsp40"; the induction was expressed especially strongly in 24 hr after the stress application.

Effect of heat shock protein 90 (Hsp90) on migration and invasion of human cancer cells in vitro.

We studied the effect of purified native heat shock protein 90 (Hsp90) from bovine and mouse brain on migration and invasion of human glioblastoma (A-172) and fibrosarcoma (HT1080) cells. Hsp90 in concentrations of 0.01-0.10 mg/ml stimulated migration and invasion of tumor cells in vitro by 20-32% (p<0.05). Polyclonal antibodies to Hsp90 blocked the Hsp90-dependent stimulation of cell invasion, which indicates specificity of the stimulating effect of extracellular Hsp90 on tumor cell invasion. Hence, extracellular Hsp90 can be considered as a promising molecular target, because its inhibition can suppress invasion and metastasizing of tumor cells.

Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Caenorhabditis elegans Hsp70-1 expresses highly in bacteria, is sufficiently soluble, and has a catalytic constant similar to Hsc70 and BiP.

Caenorhabditis elegans has been used as a model organism to study the roles of molecular chaperones in cellular processes. C. elegans heat shock protein 70-1 (CeHsp70-1) is the first of the 13-member Hsp70 family genes identified so far in the organism. The protein product of this gene, CeHsp70-1, has been shown to play an important role in conferring thermo-tolerance and longevity on C. elegans. Here, we present the results of the first work to over-express, purify and characterize the ATP hydrolyzing activity of a member of the C. elegans Hsp70s. Recombinant CeHsp70-1 was found to be highly expressed and sufficiently soluble in Escherichia coli. The protein was purified to homogeneity using a combination of nickel affinity, ion exchange and size-exclusion chromatography. Kinetic properties of the basal ATPase activity of the enzyme in a low-salt buffer were determined using a colorimetric assay. The specific activity (V(max) per mg protein), K(m) and k(cat) values obtained for CeHsp70-1 were 25 nmol/min/mg, 50 µM and 0.28 min⁻¹, respectively. The catalytic constant (k(cat)) of the protein was found to be similar to that of heat shock cognate 70 (Hsc70) and binding immunoglobulin protein (BiP). At low concentrations, CeHsp70-1 existed mostly in its monomeric form. This work provides a platform for kinetic studies of other members of the C. elegans Hsp70 molecular chaperones.

Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip).

We have developed a new approach to systematically study post-transcriptional regulation in a small number of cells. Actively translating mRNAs are associated with polysomes and the newly synthesized peptide chains are closely associated with molecular chaperones such as hsp70s, which assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones provide an anchor with which to separate actively translating mRNAs associated with polysomes from free mRNAs. Affinity capture beads were developed to capture hsp70 chaperones associated with the polysome complexes. The isolated actively translating mRNAs were used for high-throughput expression profiling analysis. Feasibility was demonstrated using an in vitro translation system with known translationally regulated mRNA transcript thymidylate synthase (TS). We further developed the approach using HCT-116 colon cancer cells with both TS and p53 as positive controls. The steady-state levels of TS and p53 mRNAs were unaltered after 5-fluorouracil treatment as assessed by real-time qRT-PCR analysis. In contrast, the protein expression and polysome-associated mRNA levels of both genes were increased. These differences in translational rate were revealed with our new approach from 500 cells. This technology has the potential to make investigation of translational control feasible with limited quantities of clinical specimens.

[Changes of heat shock proteins content in the gill epithelium cells of mussel Mytilus edulis L. depending upon the salinity of medium].

The composition and the level of heat shock (stress) proteins content were studied by the method of immunobloting in the gill epithelium cells of mussels Mytilus edulis L. from the White sea, treated by water of different salinities. Stress proteins of about 70 and 40 kDa were revealed at Western blots. After long-term (11-14 days) acclimation to 14 and 35 per thousand the level of Hsp70 in gill epithelium cells increased to compare with that in control mussels. Hsp70 induction was observed in the cells of isolated gills after salinity shock at 14 per thousand during 3 and 24 hours.

Identification of heat-shock protein 90 beta in Japanese encephalitis virus-induced secretion proteins.

Five host cellular proteins were identified in the secretion medium from Japanese encephalitis virus (JEV)-infected baby hamster kidney-21 (BHK-21) cells, including three molecular chaperones: Hsp70, GRP78 and Hsp90. Hsp90 isoforms were characterized further. Hsp90α was observed to be retained inside the nuclei, whereas Hsp90ß associated with virus particles during assembly and was released into the secretion medium upon JEV infection. The association of Hsp90ß and viral E protein was demonstrated by using sucrose-density fractionation and Western blot analysis. Moreover, JEV viral RNA replication was not affected by treatment with geldanamycin, an Hsp90 inhibitor, but impaired virus infectivity that was determined by a plaque-forming assay. Our results show that Hsp90ß, not Hsp90α, is present in the JEV-induced secretion medium and is required for JEV infectivity in BHK-21 cells.

Heat shock protein DnaK--substrate of actin-specific bacterial protease ECP32.

It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ~1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.

Inhibition of heat tolerance and nuclear import of Gts1p by Ssa1p and Ssa2p.

The Saccharomyces cerevisiae gene GTS1 is pleiotropic. GTS1 induction produces a variety of biological phenomena represented by heat tolerance. To clarify the interaction partners of Gts1p, tandem affinity purification and immunoprecipitation were performed. Ssa1p and Ssa2p, members of the 70-kDa heat-shock protein family, were identified. Co-expression of SSA1 or SSA2 inhibited Gts1p nuclear import. As compared to the wild type, the SSA1 and SSA2 double-deletion mutant showed enhancement of Gts1p-mediated heat tolerance in the stationary phase, although neither of the single deletions affected heat tolerance, irrespective of GTS1 induction. These results indicate that the heat tolerance function of Gts1p is regulated by Ssa1p and Ssa2p. Furthermore, time-dependent production of Ssa1p and Ssa2p revealed that Gts1p controls the production of Ssa1p and Ssa2p, and that the total amounts of Ssa1p and Ssa2p are important in inhibiting the unique function of Gts1p.

Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70.

Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.