Agarose Affinity Chromatography Research Articles

Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia

En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of β-phellandrene synthase, LaβPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaβPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into β-phellandrene as the major product, with K (m) and k (cat) of 6.55 μM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is produced, but were barely detectable in flowers and older leaves, where β-phellandrene is not synthesized in significant quantities. This data indicate that β-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.

Clinical significance and diagnostic value of Survivin autoantibody in non-small cell lung cancer patients

背景与目的在世界范围内，恶性肿瘤已经成为威胁人类健康的主要原因，且其发病率和死亡率居高不下。随着人们对肿瘤标志物研究的不断深入，肿瘤相关的自身抗体成为了研究热点。肺癌患者血清中Survivin自身抗体的临床意义目前还存在争议。本研究旨在探讨Survivin自身抗体在非小细胞肺癌患者血清中可能的临床应用价值。方法采用RT-PCR方法获得Survivin cDNA，构建原核表达载体pET30a(+)/Survivin，亲和层析方法纯化蛋白，SDS-PAGE电泳及Western blot鉴定，建立基于Survivin融合蛋白的间接ELISA方法，对89例健康志愿者、215例非小细胞肺癌患者以及20例肺部良性疾病患者的血清样本进行检测。结果重组Survivin融合蛋白在BL21(DE3)中以包涵体形式高效表达，间接ELISA方法检测Survivin自身抗体在非小细胞肺癌患者血清中的阳性率为19.5%，特异性为88.9%，Survivin自身抗体与非小细胞肺癌患者的肿瘤大小、远处转移间存在相关性（P < 0.05），Survivin自身抗体与CEA在非小细胞肺癌患者中联合检测的阳性率明显高于CEA与NSE、SCC、CYFRA、ProGRP联合检测的阳性率，这大大提高了非小细胞肺癌检测的敏感性。结论本研究成功构建原核表达载体pET30a(+)/Survivin，并建立检测Survivin自身抗体的间接ELISA方法，Survivin自身抗体与肺癌肿瘤大小、远处转移间的相关性及在非小细胞肺癌患者联合检测中的重要作用，为Survivin自身抗体在肺癌中的临床应用提供了线索和依据。

Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine–agarose affinity chromatography. The enzyme had a molecular mass of 23.9kDa, NH2-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50°C, whereas, with BAPNA activity was higher in a pH range of 7.5–11.5 and at 70°C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60°C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Expression and Purification of Sunflower Cotyledon Acetoacetyl CoA Thiolase Mutants

Sunflower (Helianthus annuus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of Thiolase II from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and this construct was transformed into BL‐21 E. coli that were induced for production of the Thiolase II with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble Thiolase II was purified to apparent homogeneity using Ni‐NTA agarose affinity chromatography as indicated by a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.

Cloning, mRNA expression, and recombinant expression of peptidoglycan recognition protein II gene from large yellow croaker (Pseudosciaena crocea)

Peptidoglycan recognition protein II (pglyrp2) is a type of pattern recognition receptor (PRR) that has amidase activity and is structurally conserved through evolution. However, their contributions in immune defense are different between mammal pglyrp2 and its counterpart in insects. Hitherto, fish pglyrp2 was poorly known in its structure, expression pattern and its contribution in immune defense. In present study, the pglyrp2 gene of the large yellow croaker (Pseudosciaena crocea) was cloned by RACE approach; the full-length cDNA (1842 bp) of pglyrp2 of P. crocea contains a 1446 bp open reading frame that encodes a putative protein of 482 amino acids (aa) with one 21-residue signal peptide. The pglyrp2 fusion protein and mature peptide fusion protein of P. crocea expressed by pET28a vector in the insoluble inclusion bodies (IBs) of Escherichia coli BL21 were confirmed by SDS-PAGE and subsequently purified to homogeneity by Ni-NTA agarose affinity chromatography. In addition, quantitative Real-time PCR (QRT-PCR) assays indicated that large yellow croaker pglyrp2 could be strongly expressed in liver and weakly in gonad, intestine, and stomach. Also, maternally derived pglyrp2 mRNA displayed a high level in unfertilized eggs and low expression throughout embryogenesis and yolk-sac larvae stage. Moreover, as shown in an artificial infection model, the pglyrp2 of P. crocea was confirmed to be a constitutive and inducible acute-phase protein, inducibility of which correlated with activation of anti-oxidant defense response. Thus, pglyrp2 of P. crocea was believed to play an important role in defending the eggs, bacteria recognition and activation of downstream host immune defense.

Antitumour Response of a Double Mutant of Staphylococcal Enterotoxin C2 with the Decreased Affinity for MHC Class II Molecule

Staphylococcal enterotoxin C2 (SEC2) is one of the most potent known activators of human T lymphocytes, and recombinant SEC2 shows promising clinical values, but SEC2 can cause food poisoning and toxic shock syndrome in vivo. In this study, site-directed mutagenesis has been used to introduce alanine substitutions at Phe144 and Leu45 in the molecule. The mutant genes were cloned and expressed, and the corresponding proteins were purified by nickel agarose affinity chromatography. We found that the SEC2 mutant proteins could stimulate the proliferation of human peripheral blood lymphocytes and inhibit the growth of tumour cells as native SEC2. Furthermore, flow cytometry assay showed that mSEC2(F44A, L45A) drastically reduced the ability of the toxin to bind to MHC class II. Physiological parameters revealed that mSEC2(F44A, L45A) reduced significantly rat temperature compared with native SEC2 in vivo. Our results clearly suggest that this genetically modified SEC2 protein is less toxic and justifies its further development as a new, safer antitumour superantigen to prevent SEC2 intoxication.

-D-Galactosidase from Paenibacillus thiaminolyticus catalyzing transfucosylation reactions

A genomic library of bacterial strain Paenibacillus thiaminolyticus was constructed and the plasmid DNA of the clone, containing the gene encoding beta-d-galactosidase with beta-d-fucosidase activity, detected by 5-bromo-4-chloro-3-indoxyl beta-d-galactopyranoside, was sequenced. Cells of Escherichia coli BL21 (DE3) were used for production of the enzyme in the form of a histidine-tagged protein. This recombinant fusion protein was purified using Ni-NTA agarose affinity chromatography and characterized by using p-nitrophenyl beta-d-fucopyranoside (K(m) value of (1.18 +/- 0.06) mmol/L), p-nitrophenyl beta-d-galactopyranoside (K(m) value of (250 +/- 40) mmol/L), p-nitrophenyl beta-d-glucopyranoside (K(m) value of (77 +/- 6) mmol/L), and lactose (K(m) value of (206 +/- 5) mmol/L) as substrates. Optimal pH and temperature were estimated as 5.5 and 65 degrees C, respectively. According to the amino acid sequence, the molecular weight of the fusion protein was calculated to be 68.6 kDa and gel filtration chromatography confirmed the presence of the enzyme in a monomeric form. In the following step, its ability to catalyze transfucosylation reactions was tested. The enzyme was able to catalyze the transfer of fucosyl moiety to different p-nitrophenyl glycopyranosides (producing p-nitrophenyl beta-d-fucopyranosyl-(1,3)-beta-d-fucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-glucopyranoside, p-nitrophenyl beta-d-fucopyranosyl-(1,3)-alpha-d-mannopyranoside, and p-nitrophenyl beta-d-fucopyranosyl-(1,6)-alpha-d-galactopyranoside) and alcohols (producing methyl beta-d-fucopyranoside, ethyl beta-d-fucopyranoside, 1-propyl beta-d-fucopyranoside, 2-propyl beta-d-fucopyranoside, 1-octyl beta-d-fucopyranoside, and 2-octyl beta-d-fucopyranoside). These results indicate the possibility of utilizing this enzyme as a promising tool for enzymatic synthesis of beta-d-fucosylated molecules.

Application of antibodies against nonstructural protein 2B of dengue serotype 2 virus induced by DNA immunisation or recombinant protein NS 2B immunisation in BALB/c mice

To investigate the structure and function of nonstructural (NS) protein 2B of the dengue serotype 2 virus (DV2) during infection, polyclonal antibodies (Abs) against DV2 NS2B were prepared by immunisation with NS2B protein or by DNA immunisation. The full-length NS2B gene was cloned and inserted into the prokaryotic expression vector pQE31, resulting in a vector, named pQE-NS2B, or into the eukaryotic expression vector pCAGGS-P7, resulting in the vector pCAG-NS2B. The pQE-NS2B vector was transfected into Escherichia coli JM109, and recombinant NS2B protein was obtained by Ni 2+–NTA agarose affinity chromatography. Vero cells transfected with pCAG-NS2B showed that NS2B protein can be expressed in eukaryotic cells. Finally, mice were immunised with the recombinant NS2B protein or pCAG-NS2B. Anti-NS2B sera from the immunised mice could specifically react with DV2 NS2B proteins, as visualised by fluorescence staining and Western blotting. Immunisation with NS2B protein induced a higher titre of the antibody than that induced by DNA immunisation. These data indicate that our antisera against DV2 NS2B can recognise both the natural and denatured NS2B protein. Based on these results, the polyclonal Abs could be used as a tool for studying the role of NS2B in the pathogenesis of DV2.

Optimization of expression and purification of acetoacetyl CoA thiolase from sunflower cotyledon

Sunflower (Helianthus annus L) cotyledons have two thiolase activities which have been identified previously. Thiolase I (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase II (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of a Thiolase I from an E. coli expression system. The sunflower thiolase was cloned into the pBAD‐HisB expression vector, and this construct was transformed into BL‐21 E. coli which were induced for production of the Thiolase I with 0.2% arabinose. The soluble Thiolase 1 was purified using Ni‐NTA agarose affinity chromatography with a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH 3-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione–agarose affinity chromatography and characterized by their Michaelis–Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I 50 value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.

Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R)

An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% ( 192QQ), 30.1% ( 192QR) and 44.4% ( 192RR), and 82.5% ( 55LL), 17.5% ( 55LM) and 0% ( 55MM), respectively. 192Q and 192R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V max and K m were measured using Lineweaver–Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 °C. For sarin and soman, the V max for 192Q PON1 were 3.5- and 1.5-fold higher than those for 192R PON1; and k cat/K m for 192Q PON1 were 1.3- and 2.8-fold higher than those for 192R PON1. For VX, there was little difference in V max and k cat/K m between 192Q and 192R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.

Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C

In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni(2+)-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY-TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40 degrees C and at pH 7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35 degrees C and at pH 7.5 in 100 mM Tris-HCl. As a cofactor candidate, Mg(2+) enhanced the activities of all three trehalose synthetic reactions significantly. TreY produced component C from acarbose by its proposed isomerase activity, but TreS did not. This study suggests that the mutation of treY can improve acarbose production by repressing component C production. Based on the data obtained in this study, a model for component C production in Actinoplanes sp. SN 223/29 is proposed.

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.

Isolation of mannose-binding C-type lectin from sea lamprey ( Petromyzon marinus) plasma and binding to Aeromonas salmonicida

The sea lamprey ( Petromyzon marinus) is a parasitic cartilaginous fish of the North American Great Lakes and a predator of many bony fish species of commercial importance to the fishing industry. Mannose-binding C-type lectin (MBL) was isolated by mannan–agarose affinity chromatography from sea lamprey plasma. Mannose-binding lectin has not before been identified and quantitated in the plasma of this sea lamprey species. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35 kDa and 65 kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. Amino acid composition analysis (mol%) of the purified lamprey MBL found high amounts of histidine, threonine, tyrosine and phenylalanine present when compared with three other vertebrate MBLs. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261 μg of MBL/ml of plasma. Plasma protein concentration was 40.1 mg/ml. Lamprey MBL was present then in plasma at 6.5 μg MBL/mg total protein. The sea lamprey MBL also specifically binds to mannose on the surface of the pathogen Aeromonas salmonicida. The presence of MBL in high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. This study describes the presence of MBL in sea lamprey plasma and evidence for a C-type lectin complement pathway of innate immunity.

On the association between amyloid fibrils and glycosaminoglycans; possible interactive role of Ca2+ and amyloid P-component.

We have previously reported the specific association of glycosaminoglycans (GAG) and proteoglycans (PG) with amyloid fibrils and characterized the polysaccharides directly extracted from amyloid-laden tissues. In the present study we further elucidate the association between purified amyloid fibrils and GAG/PG with special reference to those GAG/PG associated with amyloid P-component (AP) and the interactive role of Ca2+ ions. Amyloid fibrils were isolated from human hepatic AA amyloid employing water extraction with and without preceding removal of AP, an extrafibrillar protein component of all amyloids, using sodium citrate. GAG/PG co-isolated with the amyloid extracts, with and without AP, were isolated and characterized. Agarose-affinity chromatography of extracts containing AP was performed, and the GAG associated with this extrafibrillary protein were characterized as well. Several different GAG/PG populations were demonstrated in the various extracts. The abolition of calcium-dependent binding markedly influenced the amount of GAG/PG recovered in the fibril extracts, as well as the total amount of amyloid material obtained. Thus, it seems that calcium plays an important role in the association between the fibrils and the sugar moieties, and that a significant fraction of the GAG found in amyloid exhibits a Ca(2+)-dependent fibril-GAG interaction. No significant difference in the proportion between galactosaminoglycans and glucosamines was, however, disclosed when the two extraction protocols were compared, suggesting that no particular GAG species has a higher affinity for the fibrils themselves. Both dermatan/chondroitin sulphate and heparan sulphate identified in the present study exhibited a Ca(2+)-dependent interaction with AP, supporting previous findings. However, the amyloid-associated galactosaminoglycans found, especially the large PG appearing in small amounts, seemed to have a higher affinity for the extrafibrillar AP than the other GAG.

Production of D-myo-inositol(1,2,4,5,6)pentakisphosphate using alginate-entrapped recombinant Pantoea agglomerans glucose-1-phosphatase

ABSTRACT The glucose-1-phosphatase encoding gene (agp) of Pantoea agglomerans was sequenced and heterologously expressed in Escherichia coli. The enzyme showed very high homology to periplasmatic glucose-1-phosphatases of other members of the Enterobacteriaceae family. It was isolated from transformed Escherichia coli cells in a single step in high yields (32.3 ± 1.2 mg per litre of culture) by Ni-NT agarose affinity chromatography to >95% purity as calculated from specific activity determinations. The purified glucose-1-phosphatase was entrapped in alginate beads with an entrapment efficiency of >80%. Temperature stability was enhanced as a consequence of entrapment, whereas pH dependence of enzyme activity was not affected. Maximum catalytic activity of entrapped glucose-1-phosphatase was found at 70°C, whereas the free enzyme exhibited maximal activity at 60°C. A single pH optimum at pH 4.5 was determined for the free and the entrapped enzyme. Kinetic parameters for the hydrolysis of sodium phytate were found to be affected by entrapment. They were determined to be K

Isolation of new pregnancy-associated glycoproteins from water buffalo ( Bubalus bubalis) placenta by Vicia villosa affinity chromatography

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo ( Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

The second glutamic acid in the C-terminal CRD affects the carbohydrate-binding properties of recombinant galectins of Haemonchus contortus

The effects of the second glutamic acid (E) in the C-terminal CRDs on the hemagglutination and lactose-binding characteristics of the recombinant galectins of nematode Haemonchus contortus were observed using two isoforms of recombinant galectins as models, and the sugar-binding abilities of the N-terminal and C-terminal CRDs of the galectins were also compared. The second E in the CRD, WGNEER, of Hco–GAL-m was mutated to glycine acid (G) and resulted in a recombinant galectin (MG mutate) with a CRD of WGNEGR, identical to that of Hco–GAL-f. The G in Hco–GAL-f CRD, WGNEGR, was mutated to E and produced a recombinant galectin (FE mutate) equal to that of Hco–GAL-m. At the same time, the CRDs of the N-terminal (FNh,MNh) and C-terminal (FCh,MCh) of Hco–GAL-f, Hco–GAL-m were amplified by PCR. The abilities of carbohydrate binding and hemagglutination of the four galectins and the four CRDs were analysed, respectively, by α-lactose–agarose affinity chromatography and hemagglutination assay. The results showed that Hco–GAL-m and FE mutate bound effectively to α-lactose–agarose compared to Hco–GAL-f and MG mutate, which almost could not bind to the conjugate column. The hemagglutinating abilities of the Hco–GAL-m and FE mutate to human B type red blood cells were similar and were nearly two times higher than that of the Hco–GAL-f and MG mutate. The hemagglutinating ability of the MCh was five times to that of the MNh and FNh and almost two times to that of the FCh. The binding ability of the MCh and FCh were significantly reduced compared to that of the Hco–GAL-m and FE mutate, but still remained. As for the MNh and FNh, no elution peak was observed in the lactose–agarose affinity chromatography. These results suggested that the second amino acid E in the C-ternminal CRD motif of H. contortus galectin was involved in carbohydrate binding and hemagglutination, and C-terminal CRDs had stronger carbohydrate ability than N-terminal CRDs.

Expression and bioactivity of recombinant segments of human perforinThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

The aim of the study was to prepare an active recombinant human perforin by comparing 5 candidate segments of human perforin. Full-length perforin, MAC1 (28-349 aa), MAC2 (166-369 aa), C-100, and N-60 of human perforin were selected as candidate active segments and designated, respectively, HP1, HP2, HP3, HP4, and HP5. The target genes were amplified by PCR and the products were individually subcloned into pGEM-T. The genes for HP1, HP2, HP3, and HP5 were subcloned into pET-DsbA, whereas pET-41a (+) was used as the expression vector of HP4. The fusion proteins were expressed in Escherichia coli BL21pLysS(DE3) and purified using nickel nitrilotriacetic acid (NTA) agarose affinity chromatography. The hemolysis microassay was used as an activity assay of fusion protein. From this study, we obtained the recombinant plasmids pGEM-T-HP1, -HP2, -HP3, -HP4 and -HP5, consisting of 1600, 960, 600, 300bp, and 180, respectively. From these recombinant plasmids, expression plasmids were successfully constructed and expressed in E. coli BL21pLysS(DE3). The resultant fusion proteins, affinity purified using Ni-NTA, were approximately 80, 58, 45, 44, and 30 kDa, respectively. The recombinant proteins were assayed for activity on hemolysis. HP2 and HP5 were the only recombinant proteins that were active in hemolysis, and the hemolytic function was concentration dependent. These results demonstrate that active recombinant forms of perforin can be synthesized in a prokaryote model. The recombinant N-60 and MAC1 (28-349 aa) of human perforin have the function of forming pores. Our study provides the experimental basis for further investigation on the application of perforin.