Sodium Dodecyl Sulfate-polyacrylamide Gel Eletrophoresis Research Articles

A novel membrane based process to isolate recombinant human chemokine receptor CCR3 produced in Escherichia coli

The isolation of recombinant human chemokine receptor CCR3, which fused to maltose binding protein (MBP), has been conducted using ultrafiltration with 300kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on MBP-CCR3 stability and transmission were quantified using dot blot analysis and parameter scanning ultrafiltration respectively. These conditions included solution pH, ionic strength, stirring speed, permeate flux, and detergent (Fos choline-14) concentration. Under optimized conditions, the MBP-CCR3 purity obtained in the retentate was about 96% and the recovery of MBP-CCR3 was close to 89% after ultrafiltration. The resulting MBP-CCR3 product was then analyzed by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. To our knowledge, this is the first paper of the isolation of a G protein coupled receptor (GPCR) using ultrafiltration alone.

Biochemical Properties of Pelagic Fish Proteins as Affected by Isolation Methods and Gel Properties by Heating Methods

The biochemical and gel properties of Pacific sardine and Pacific mackerel were characterized as affected by preparation and cooking methods. Four to eight times more salt soluble proteins were extracted from water-washed paste than fish protein isolate (FPI) paste. Higher total sulfhydryl content was measured in FPI, indicating the exposure of sulfhydryl groups during alkaline extraction. Comparing gel properties based on two cooking methods (slow and fast), the two pelagic fish proteins performed quite differently. Heating rate did not differentiate between surimi and FPI gels from sardine. However, mackerel exhibited higher texture values when using the fast cooking method, indicating the presence of high levels of proteolytic enzymes. Water-washed surimi gels were whiter than FPI for both species. Water retention ability appeared to be higher with mackerel than sardine, regardless of isolation and cooking method. Sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) also supported a difference in processing chemistry and thermal behavior between two methods for protein isolation and cooking, respectively.

Expression of Kaposi’s sarcoma-associated herpesvirus ORFK8.1 and its preliminary diagnostic application

The ORFK8.1 of Kaposi’s sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf K8.1 was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). The fusion protein was purified by chromatyography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). SDS-PAGE showed that a protein of 26 kDa was visualized as expected. A western blot assay was established to analyze the immunogenicity of purified recombinant ORFK8.1 protein. The optimal condition of the recombinant ORFK8.1 ELISA assay was confirmed: the concentration of antigen was 5 μg/mL, the dilution of serum was 1:200. We used the ELISA method to investigate the recombinant ORF K8.1 protein’s specificity, the data showed that the specificity of ORF K8.1 to detect KSHV was 100%. At the same time, 560 sera samples from Hubei province were detected by using ORFK8.1 ELISA to investigate KSHV seroprevalence in this region. The KSHV seroprevalence in Hubei province is shown to be 6.80%.

A two-dimensional protein map of Caenorhabditis elegans

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12 % sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

A two-dimensional protein map of Caenorhabditis elegans.

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

Expression and characterization of recombinant human-derived Pneumocystis carinii dihydrofolate reductase.

Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the rat-derived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K(m) values for dihydrofolate and NADPH were 2.7 +/- 0.3 and 14.0 +/- 4.3 microM, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K(i) values of 0.28 +/- 0.08 and 0.065 +/- 0.005 microM, respectively, while trimetrexate and methotrexate were potent inhibitors, with K(i) values of 0.23 +/- 0.03 and 0.016 +/- 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for human-derived P. carinii DHFR.

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection.

The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.

Seed Storage Protein Profiles of Seven Indian Wheat (Triticum aestivum L.) Varieties

Seed storage protein profiles, Including triticin, gliadin and glutenin, of seven Indian wheat varieties were Investigated using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (50S-PAGE). A strategy was developed for half-seed analysis of the three seed storage protein classes. The results are presented In the form of a key for Identification of these varieties based on their seed protein composition. A minimum of 10 seeds of each variety were analysed to check for possible intravarietal heterogeneity, and all the varieties except HUW-12 were homogeneous. The HUW-12 was a mixture of two ‘biotypes’ with respect to the low molecular weight (LMW) glutenin subunits coded by the Glu-A3 locus. The varieties were also analysed for the presence of rye chromatin using a rye-specific repetitive DNA probe (pAW-161), and two of the recently released varieties HUW-206 and HUW-318 were found to possess rye chromatin. Based on the DNA dot-blot results, presence of rye secalins and the absence of chromosome 1 B-speclflc gliadlns It was concluded that these two varieties possess 1BL-1RS wheat-rye translocation. Both of these varieties also have high molecular weight (HMW) glutenin subunits 5 + 10 which may be necessary for compensating the loss of dough strength associated with the wheat-rye translocations.

An oat coleoptile wall protein that induces wall extension in vitro and that is antigenically related to a similar protein from cucumber hypocotyls

Plant cell walls expand considerably during cell enlargement, but the biochemical reactions leading to wall expansion are unknown. McQueen-Mason et al. (1992, Plant Cell 4, 1425) recently identified two proteins from cucumber (Cucumis sativus L.) that induced extension in walls isolated from dicotyledons, but were relatively ineffective on grass coleoptile walls. Here we report the identification and partial characterization of an oat (Avena sativa L.) coleoptile wall protein with similar properties. The oat protein has an apparent molecular mass of 29 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Activity was optimal between pH 4.5 and 5.0, which makes it a suitable candidate for “acid growth” responses of plant cell walls. The oat protein induced extension in walls from oat coleoptiles, cucumber hypocotyls and pea (Pisum sativum L.) epicotyls and was specifically recognized by an antibody raised against the 29-kDa wall-extension-inducing protein from cucumber hypocotyls. Contrary to the situation in cucumber walls, the acid-extension response in heat-inactivated oat walls was only partially restored by oat or cucumber wall-extension proteins. Our results show that an antigenically conserved protein in the walls of cucumber and oat seedlings is able to mediate a form of acid-induced wall extension. This implies that dicotyledons and grasses share a common biochemical mechanism for at least part of acid-induced wall extensions, despite the significant differences in wall composition between these two classes of plants.

Purification and characterizations of β-ketoacyl-[acyl-carrier-protein] reductase, β-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-carrier-protein] reductase from Spinacia oleracea leaves

β-Ketoacyl-[acyl-carrier-protein] (ACP) reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase have been purified to homogeneity from extracts of spinach leaves. Based on sodium dodecyl sulfate-polyacrylamide gel eletrophoresis studies, the monomeric molecular weights of the β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase were 24,200, 19,000, and 32,500, respectively, and by gel filtration, their molecular weights were 97,000, 85,000, and 115,000, respectively, suggesting that these three enzymes exist as tetramers. The β-ketoacyl-ACP reductase, the β-hydroxyacyl-ACP dehydrase, and the enoyl-ACP reductase contained two, one, and two cystein residues per monomer. β-Ketoacyl-ACP reductase preferably utilized NADPH as the reductant, whereas enoyl-ACP reductase was absolutely specific to NADH. β-Ketoacyl-ACP reductase reversibly catalyzed the reduction of acetoacetyl-ACP to d-β-hydroxybutyryl-ACP and β-hydroxyacyl-ACP dehydrase catalyzed the dehydration of d-β-hydroxyacyl-ACP to 2-enoyl-ACP. Both β-hydroxyacyl-ACP dehydrase and enoyl-ACP reductase were active with 2-enoyl-ACPs having chain lengths from C 4 to C 16, with 2-hexenoyl-ACP and 2-octenoyl-ACP being the most effective substrate. CoA esters served as substrates with the β-ketoacyl-ACP reductase and the enoyl-ACP reductase but were inert with β-hydroxyacyl-ACP dehydrase. These enzymes were inhibited by p-chloromercuribenzoate but not by N-ethylmaleimide.