Affinity Chromatography Purification Research Articles

Wiskott-Aldrich Protein Is Phosphorylated by NPM-ALK and Regulates the Proliferation and Invasion of Anaplastic Large Cell Lymphoma

Abstract 1535 Introduction:Anaplastic large cell lymphoma (ALCL) is a subtype of mature T cell lymphoma. The NPM-ALK fusion protein is expressed in 80% of pediatric ALCL and arises from a chromosomal translocation t(2;5)(p23;q35). NPM-ALK is a constitutively active oncogenic tyrosine kinase that regulates the function of numerous proteins involved in apoptosis and cell cycle progression. In order to better understand the mechanisms of NPM-ALK mediated oncogenesis, we utilized an unbiased mass spectrometry –based phosphoproteomic approach to identify novel downstream signaling targets of the oncogenic NPM-ALK. Approach:The phosphoproteome was enriched using a two-step procedure including immobilized metal affinity chromatography (IMAC) and phospho-tyrosine immunoaffinity purification and subsequently analyzed by liquid chromatography and high mass accuracy tandem mass spectrometry (LC-MS/MS). Western blot analysis with phospho-specific antibodies and in vitro kinase assays were used to validate the MS results. Stable lentiviral transduction of shRNA vectors was used to evaluate the functional consequences. Cell proliferation was measured using the WST-1 assay. Effect on transformation capacity was assessed with colony formation assay using methyl-cellulose agar. Invasive properties were assessed using collagen based transwell assays and adhesion was evaluated by attachment onto fibronectin substratum. Results:Phosphoproteomic analysis identified several proteins involved in actin dynamics to be regulated by ALK tyrosine kinase activity. These include WASP, CRKL, NCK1, ARP2/3 and ITSN2. Pathway network analysis situated WASP at the center of the actin network. Western blot analysis demonstrated that phosphorylation of WASP at Y290 is regulated by NPM-ALK. Expression of wild type NPM-ALK increased the phosphorylation of WASP Y290 in 293T cells compared to a kinase-defective K210R mutant. In vitro kinase assays demonstrated that NPM-ALK phosphorylates WASP at Y290 as well as a novel residue Y102. The interaction between NPM-ALK and WASP was demonstrated using immunoprecipitation of co-transfected 293T cells as well as in ALCL cell lines. Because Y102 residue of WASP is located in the domain involved in interaction with its chaperone WIP, we evaluated whether its phosphorylation regulates the interaction. The interaction of WASP and WIP was decreased when Y102 was phosphorylated as demonstrated by immunoprecipitation of phospho-mutants of WASP (Y102F and Y102E). Surface plasmon resonance studies with recombinant WT or phosphomimetic (Y102E) WASP and WIP confirmed these observations. Furthermore, cycloheximide chase experiments demonstrated that phosphorylation of WASP at Y102 decreased it protein stability. Stable WASP knockdown in SUDHL1 cells led to reduced adhesion to fibronectin (21% ± SEM 0.4% vs 13% ± SEM 0.6%, p<0.01) and invasion through collagen (68% ± SEM 16% vs 12% ± SEM 3%, p<0.05). Interestingly, stable knock down of WASP resulted in increased proliferation (0.79 RFU ± SEM 0.07 vs 0.54 RFU ± SEM 0.019, p<0.01) and colony formation (58 colonies ± SEM 1.2 vs 26 colonies ± SEM 3.3, p< 0.01) of SUDH1L cells. Conclusions:Our results show that NPM-ALK regulates the phosphorylation of a network of proteins involved in actin dynamics, one of which is WASP. NPM-ALK directly phosphorylates WASP at residues Y290 and Y102 and thereby regulates WASP function in actin dynamics critical for cell adhesion, invasion and proliferation. Disclosures:No relevant conflicts of interest to declare.

Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66-68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin-methotrexate (CDDP-MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66-68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66-68kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.

Affinity chromatographic purification of human immunoglobulin M from human B lymphocyte cell culture supernatant

Compared to immunoglobulin G purification with extensively studied affinity ligands such as protein A and protein G, little work has been done on affinity chromatographic purification of immunoglobulin M. Hexamer peptide ligand HWRGWV, previously shown to bind specifically to the Fc fragment of IgG, also demonstrated potential for IgM purification. This study presents further characterization and investigation of this ligand for its potential for purification of IgM. Different running conditions were employed in order to improve the recovery and purity of IgM. The final recovery and purity of the antibody is feedstock dependent, but can reach levels of both recovery and purity as high as 95%. The dependence of the recovery and purity on total loading amount and initial IgM concentration were investigated and discussed. Although relatively low dynamic binding capacities (DBC) in the range of 4.6–13.1mg IgM/mL resin at linear flow rates from 173 to 35cm/h were obtained for IgM compared to IgG because of the large molecular weight of IgM, the DBC value of HWRGWV for IgM is much greater than protein-based IgM affinity ligands found in the literature and is competitive with current commercially available affinity ligands, such as KAPTIVE-M, CaptureSelect IgM and Ultralink Immobilized Mannan Binding Protein.

Viral resistance and immune responses of the shrimp Litopenaeus vannamei vaccinated by two WSSV structural proteins

Although adaptive immunity is believed to exist only in higher vertebrates, recent studies showed the ability to vaccinate shrimp and other crayfish against white spot syndrome virus (WSSV). This study attempted to establish parameters of vaccination coordinated with subsequent viral challenge to gain insights into the mechanisms of the protective response of penaeid shrimp. Two WSSV envelope proteins, VP28 and VP36B, were used as subunit vaccines expressed in Escherichia coli followed by histidine-tag affinity chromatographic purification. Shrimp vaccinated with the recombinant WSSV proteins and challenged with diluted WSSV inocula were intramuscularly injected in order to give a precise load. Results of the viral challenge trials showed complete survival in the rVP28 group in contrast to the rVP36B and PBS groups which exhibited 100% mortality. But this effective protection was exclusively induced from a combination of a higher dosage of rVP28 and a lower viral challenge pressure. The innate immune parameters analyzed among the three groups revealed that rVP28-treated shrimp showed the highest activity level (p<0.05) of phenoloxidase and superoxide dismutase during the entire period of 7 days post-vaccination. But there were no significant differences (p>0.05) in mRNA abundances of the Down syndrome cell adhesion molecule (Dscam) among all groups. In addition, total hemocyte counts significantly decreased in shrimp treated with the recombinant viral proteins compared to the PBS group. These results indicated that the existence of structure- and dose-dependent protective responses and the elevated innate immunity in shrimp following a protein-based vaccination might be responsible for conferring resistance against WSSV.

High‐level production of soluble pyrroloquinoline quinone‐dependent glucose dehydrogenase in Escherichia coli

Pyrroloquinoline quinone‐dependent glucose dehydrogenase (EC1.1.5.2, PQQGDH) has attracted progressive attention due to its application in glucose detection in clinic diagnosis and industrial bioprocess controls. To satisfy its increasing demand, improvement of PQQGDH production derived from Acinetobacter calcoaceticus L.M.D. 79.41 in recombinant Escherichia coli is necessary and is therefore the focus of the current study. Different carbon sources as well as induction conditions were investigated for overexpression of soluble PQQGDH. The results indicate that the target protein was optimally produced with 20 g/L glucose as the substrate. Moreover, the highest expression level (1530 kU/L) was achieved by a novel two‐temperature cultivation strategy in the 10‐L fermentor. This presents a sixfold improvement over previously reported values. After Ni‐NTA affinity chromatography purification, high‐purity enzyme with the specific activity of 5811 U/mg was obtained with a purification yield of 55%. The purified recombinant PQQGDH showed thermal stability and substrate specificity as the native enzyme. In summary, this work provides an alternative production process to overexpress PQQGDH and shows high applicability for large‐scale production of this important glucose dehydrogenase.

Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system

The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.

Identification of a potential tumor differentiation factor receptor candidate in prostate cancer cells

Tumor differentiation factor (TDF) is a pituitary protein that is secreted into the bloodstream and has an endocrine function. TDF and TDF-P1, a 20-residue peptide selected from the ORF of TDF, induce differentiation in human breast and prostate cancer cells, but not in other cells. TDF has no known mechanism of action. In our recent study, we identified heat shock 70 kDa proteins (HSP70s) as TDF receptors (TDF-Rs) in breast cancer cells. Therefore, we sought to investigate whether TDF-R candidates from prostate cancer cells are the same as those identified in breast cancer cells. Here, we used TDF-P1 to purify the potential TDF-R candidates by affinity purification chromatography from DU145 and PC3 steroid-resistant prostate cancer cells, LNCaP steroid-responsive prostate cancer cells, and nonprostate NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. We identified the purified proteins by MS, and validated them by western blotting, immunofluorescence microscopy, immunoaffinity purification chromatography, and structural biology. We identified seven candidate proteins, of which three were from the HSP70 family. These three proteins were validated as potential TDF-R candidates in LNCaP steroid-responsive and in DU145 and PC3 steroid-resistant prostate cancer cells, but not in NG108 neuroblastoma and BLK CL.4 fibroblast-like cells. Our previous study and the current study suggest that GRP78, and perhaps HSP70s, are strong TDF-R candidates, and further suggest that TDF interacts with its receptors exclusively in breast and prostate cells, inducing cell differentiation through a novel, steroid-independent pathway.

Plasmodium yoelii blood-stage antigens newly identified by immunoaffinity using purified IgG antibodies from malaria-resistant mice

As the search for an effective human malaria vaccine continues, understanding immune responses to Plasmodium in rodent models is perhaps the key to unlocking new vaccine strategies. The recruitment of parasite-specific antibodies is an important component of natural immunity against infection in blood-stage malaria. Here, we describe the use of sera from naturally surviving ICR mice after infection with lethal doses of Plasmodium yoelii yoelii 17XL to identify highly immunogenic blood-stage antigens. Immobilized protein A/G was used for the affinity-chromatography purification of the IgGs present in pooled sera from surviving mice. These protective IgGs, covalently immobilized on agarose columns, were then used to isolate reactive antigens from whole P. yoelii yoelii 17XL protein extracts obtained from the blood-stage malaria infection. Through proteomics analysis of the recovered parasite antigens, we were able to identify two endoplasmic reticulum lumen proteins: protein disulfide isomerase and a member of the heat shock protein 70 family. Also identified were the digestive protease plasmepsin and the 39kDa-subunit of eukaryotic translation initiation factor 3, a ribosome associated protein. Of these four proteins, three have not been previously identified as antigenic during blood-stage malaria infection. This procedure of isolating and identifying parasite antigens using serum IgGs from malaria-protected individuals could be a novel strategy for the development of multi-antigen-based vaccine therapies.

New IMAC media enabling purification of histidine‐tagged proteins directly from eukaryotic cell culture supernatants

Immobilized metal affinity chromatography (IMAC) purification of secreted histidine‐tagged proteins in eukaryotic cell culture supernatants is often problematic. Incompatibility with the cell culture media appears as stripping of the immobilized metal ions required for binding of target proteins. Due to low target protein concentration in cell culture supernatants, large sample volumes are often needed, aggravating the stripping effect.In this study, purifications of histidine‐tagged proteins were performed using two novel Sepharose™ based IMAC media; Ni Sepharose excel and magnetic His Mag Sepharose excel. Both media have a new type of chelating ligand with exceptionally strong binding of nickel ions. Data showing successful purification of histidine‐tagged proteins from CHO cell culture supernatants will be presented.Furthermore, the characteristics of new media enabled purification of target protein from insect cell culture supernatants. The purification was easily scaled up from 20 μl His Mag Sepharose excel beads to 1 mL pre‐packed columns.

Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein

Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus.

Production, purification and cytotoxity of soluble human Fas ligand expressed by Escherichia coli and Dictyostelium discoideum

Human Fas ligand (hFasL) is a type II membrane protein that induces apoptosis in the Fas-bearing cells. Its special biological activity has the potential for the therapeutic use as an anti-cancer agent directed at enhancing apoptosis in tumor cells. In this study Escherichia coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amounts. An expression vector for hFasL production in E. coli was constructed based on plasmid pET32a(+). By cultivation of the hFasL-producing E. coli clone on LB medium and induction with IPTG, a hFasL concentration of 1.0mgL−1 was achieved. D. discoideum strain AX3-hFasL-H was cultured in a conventional stirred bioreactor on an improved synthetic medium using a simple fed-batch strategy, and cell densities of up to 8.3×107cells/mL and a maximum hFasL concentration of 420μg/L were obtained. Using Ni-NTA affinity chromatography purification, two kinds of recombinant hFasLs from E. coli and D. discoideum were purified with a purity of 94% and 90%, respectively. They showed similar biological activities in inducing apoptosis in Fas-expressing cells.

Identification of Potential Tumor Differentiation Factor (TDF) Receptor from Steroid-responsive and Steroid-resistant Breast Cancer Cells

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04–0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.

English

Streptokinase (SK) is a therapeutically important thrombolytic agent. Cardiovascular disease is the first cause of adult death worldwide. In Egypt about 13% of the population die every year due to ischemic heart disease. In spite of this fact, there is no local production of cardiovascular therapeutics. We reported for the first time the expression of a recombinant SK from a local&nbsp;Streptococcus&nbsp;strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from&nbsp;Streptococcus&nbsp;sp. isolated from Egyptian pharyngitis patients was obtained. The isolated strain was partially identified using 16S rDNA sequencing and namedStreptococcus&nbsp;sp.&nbsp;SalMarEg. It was found to be phylogenetically related toStreptococcus&nbsp;pyogenes.&nbsp;Analysis of the obtained sequence showed high similarity with other SK genes. The protein expression in a prokaryotic system obtained a 47-kDa SK protein that could be purified using a single-step his-tagged affinity purification chromatography, with nearly 80% recovery. The clot lytic activities of both recombinant and commercial SK were similar, thus giving the basis to scale up this SK product in order to evaluate the possibilities of its commercialization in local and/or regional markets. &nbsp; Key words:&nbsp;Streptokinase,&nbsp;Streptococcus SalMarEg,&nbsp;thrombolytic agent,heterologous&nbsp;expression.

Affinity purification of a chimeric nicotinic acetylcholine receptor in the agonist and antagonist bound states

Nicotinic acetylcholine receptors (nAChRs) form ligand-gated ion channels that mediate fast signal transmission at synapses. These receptors are members of a large family of pentameric ion channels that are of active medical interest. An expression system utilizing a chimerical construct of the N-terminal extracellular ligand binding domain of alpha7 type nAChR and the C-terminal transmembrane portion of 5HT3 type receptor resulted high level of expressions. Two ligand affinity chromatography purification methods for this receptor have been developed. One method relies on the covalent immobilization of a high affinity small molecule alpha7 nAChR agonist, (R)-5-(4-aminophenyl)-N-(quinuclidin-3-yl) furan-2-carboxamide, and the other uses mono biotinylated alpha-bungarotoxin, an antagonist, that forms a quasi-irreversible complex with alpha7 nAChR. Detergent solubilized alpha7/5HT 3 chimeric receptors were selectively retained on the affinity resins and could be eluted with free ligand or biotin. The proteins purified by both methods were characterized by gel electrophoresis, mass spectra, amino acid composition analysis, and N-terminal sequence determination. These analyses confirmed the isolation of a mature alpha7/5HT 3 receptor with the signal peptide removed. These results suggest a scalable path forward to generate multi-milligram amounts of purified complexes for additional studies including protein crystallization.

IMPROVEMENT OF PURIFICATION OF TRYPSIN INHIBITOR FROM WILD SOYBEAN (GLYCINE SOJA SIEB. &amp; ZUCC.) USING CHITOSAN RESIN-IMMOBILIZED TRYPSIN

Chitosan resin-trypsin was prepared for one-step affinity purification of trypsin inhibitor (TI) from a wild-type soybean (Glycine soja Sieb. & Zucc.). The influences of several parameters (cross-linking, chemical modifying and activating agent) on the chitosan resin-trypsin activity were investigated. The results showed that the chitosan resin-trypsin composite demonstrated considerable trypsin activity at levels of, 3.6% glutaraldehyde (cross-linking agent), 0.06 mol/L NaBH4 (chemical modifying agent) and 15% epichlorohydrin (activating agent). Furthermore, the trypsin activity assay indicated that chitosan resin-trypsin could tolerate relatively high temperature (65C) and wide pH range (5.0–9.0), compared with free trypsin (45C, pH 6.0–8.0). With chitosan resin-trypsin as matrix on affinity chromatography column, a Bowman-Birk trypsin inhibitor from a wild soybean was purified (adsorption capacity of chitosan resin-trypsin for TI was about 1.33 mg/g wet matrix), with homogeneous MW of 8.2 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The affinity chromatography purification method described for TI was 279-fold with a 58.3% recovery rate of TI, much improved over established procedures. PRACTICAL APPLICATIONS The use of chitosan resin as a support for enzymes in affinity chromatography has advantages over other materials: low cost, good stability and versatility. The covalent immobilization of trypsin on chitosan resin permits easy recovery and reuse of the enzyme, and produces a quick and effective tool to isolate the Bowman-Birk trypsin inhibitor form wild soybean. This method could be a competitive choice for large-scale purification of this functional molecule.

Substrate and Metal Ion Promiscuity in Mannosylglycerate Synthase

The enzymatic transfer of the sugar mannose from activated sugar donors is central to the synthesis of a wide range of biologically significant polysaccharides and glycoconjugates. In addition to their importance in cellular biology, mannosyltransferases also provide model systems with which to study catalytic mechanisms of glycosyl transfer. Mannosylglycerate synthase (MGS) catalyzes the synthesis of α-mannosyl-D-glycerate using GDP-mannose as the preferred donor species, a reaction that occurs with a net retention of anomeric configuration. Past work has shown that the Rhodothermus marinus MGS, classified as a GT78 glycosyltransferase, displays a GT-A fold and performs catalysis in a metal ion-dependent manner. MGS shows very unusual metal ion dependences with Mg(2+) and Ca(2+) and, to a lesser extent, Mn(2+), Ni(2+), and Co(2+), thus facilitating catalysis. Here, we probe these dependences through kinetic and calorimetric analyses of wild-type and site-directed variants of the enzyme. Mutation of residues that interact with the guanine base of GDP are correlated with a higher k(cat) value, whereas substitution of His-217, a key component of the metal coordination site, results in a change in metal specificity to Mn(2+). Structural analyses of MGS complexes not only provide insight into metal coordination but also how lactate can function as an alternative acceptor to glycerate. These studies highlight the role of flexible loops in the active center and the subsequent coordination of the divalent metal ion as key factors in MGS catalysis and metal ion dependence. Furthermore, Tyr-220, located on a flexible loop whose conformation is likely influenced by metal binding, also plays a critical role in substrate binding.

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca 2+ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL–CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL–CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.

Inhibition of angiotensin I-converting enzyme by wheat gliadin hydrolysates

A tryptic gliadin hydrolysate was fractionated into peptide fractions, which were assigned to either the central domain (CD) or terminal domains (TD) of gliadins. The domains were expected to contain amino acid (AA) sequences which, when released from the parent protein, inhibit the angiotensin I-converting enzyme (ACE), which plays a key role in regulating blood pressure. A proline (Pro) poor TD related fraction, containing the smallest peptides, showed the highest ACE inhibitory activity (IC 50 = 0.33 mg/ml). Additional peptidases were selected based on their in silico predicted ability to release ACE inhibitory peptides. Further hydrolysis of the tryptic hydrolysate fractions with thermolysin, Clarex, Alcalase and Esperase increased ACE inhibitory activities. Immobilised Ni 2+-ion affinity chromatography (IMAC) purification of a TD related peptide fraction obtained by sequential hydrolysis with trypsin and thermolysin yielded a fraction with an IC 50 value of 0.02 mg/ml. This IMAC fraction was enriched in histidine and hydrophobic AA (Pro, Val, Ile, Leu and Phe).

High-level soluble expression of recombinant human manganese superoxide dismutase in Escherichia coli, and its effects on proliferation of the leukemia cell

Manganese superoxide dismutase (Mn-SOD) is one of the major enzymes responsible for the defense against oxidative damage due to reactive oxygen species (ROS) in the mitochondria. The present study aimed to produce and evaluate the genetically engineered manganese superoxide dismutase protein. A recombinant plasmid containing DNA segment coding Mn-SOD protein was transformed into Escherichia coli ( E. coli) Rosetta-gami strain, for expression. After induction with IPTG, an expected molecular mass of 25 kDa was detected by SDS–PAGE. After Ni–NTA affinity chromatography purification, the purity rate came up to 95%. UV spectroscopy data for our preparations indicated that a peak at 275 nm existed in the spectrum. SOD activity assay showed that the activity of the rhMn-SOD was 1890.9 U/mg. The ORAC level of rhMn-SOD was 151492.2 uM Trolox equiv/mg. Furthermore, in vitro bioacitivity assay indicated that the rhMn-SOD protein can inhibit the proliferation of the leukemia K562 cells.