Identification of a retina-specific MEKA protein as a 33 K protein

A photoreceptor-specific MEKA protein was purified from bovine retinal soluble fraction. The purified sample was eluted as a single peak of 74 kDa protein from a Superose column, which was dissolved into three components, MEKA protein (32 kDa), beta-(36 kDa) and gamma-(10 kDa) subunits of transducin on a SDS-PAGE. From several lines of evidence, we concluded that MEKA protein is identical with a 33k phosphoprotein reported by Lee et al (1).     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.     

Purification of a Candida albicans germ tube specific antigen

Abstract In a previous work, Marot-Leblond et al. identified a Candida albicans germ tube-specific antigen by the use of a monoclonal antibody (mAb 3D9.3). In the present report, we used a two-step procedure to obtain a purified preparation of this antigen from a Zymolyase extract of Candida albicans germ tubes. The extract was first fractionated by gel filtration chromatography. The immunoreactive fractions were pooled, and the 3D9.3 antigen was further purified by hydrophobic interaction chromatography using a Phenyl-superose column. Analysis by SDS-PAGE, immunoblotting and Concanavalin A staining, revealed a single, polydisperse band ranging from 110 to 170 kDa. The antigen was purified 126-fold by protein content and 16.4-fold by carbohydrate content. Recovery of the antigen was 6.8% following the two-step purification.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2.

A novel ethylene-forming enzyme that catalyses the formation of ethylene from 2-oxoglutarate was purified from a cell-free extract of Pseudomonas syringae pv. phaseolicola PK2. It was purified about 2800-fold with an overall yield of 53% to a single band of protein after SDS-PAGE. The purified enzyme had a specific activity of 660 nmol ethylene min-1 (mg protein)-1. The molecular mass of the enzyme was approximately 36 kDa by gel filtration and 42 kDa by SDS-PAGE. The isoelectric point and optimum pH were 5.9 and ca. 7.0-7.5, respectively. There was no homology between the N-terminal amino acid sequence of the ethylene-forming enzyme of Ps. syringae pv. phaseolicola PK2 and the sequence of the ethylene-forming enzyme of the fungus Penicillium digitatum IFO 9372. However, the two enzymes have the following properties in common. The presence of 2-oxoglutarate, L-arginine, Fe2+ and oxygen is essential for the enzymic reaction. The enzymes are highly specific for 2-oxoglutarate as substrate and L-arginine as cofactor. EDTA, Tiron, DTNB [5,5'-dithio-bis(2-nitrobenzoate)] and hydrogen peroxide are all effective inhibitors.     

Monoclonal antibodies against the haemolysin of Vibrio vulnificus

The extracellular haemolysin produced by Vibrio vulnificus strain FCC was partially purified from the culture supernate by sequential ammonium sulphate precipitation, gel filtration with Sepharose 4B, and DEAE-Sephacel ion-exchange column chromatography. Using this semi-purified haemolysin as the antigen, several monoclonal antibodies (MAbs) were established; they were all of the IgG2b class with lambda light chains. One representative MAb, 6F8D, completely neutralized the haemolytic activity and mouse lethal activity of extracellular toxin(s). In immunoblotting analysis of the peptides of the semi-purified haemolysin separated by SDS-PAGE, this MAb reacted, in particular, with a 36 kDa peptide. These findings suggest that the haemolysin is probably identical to the lethal toxin in the culture supernate of V. vulnificus strain FCC, which contained the 36 kDa peptide.     

N-Acetyl-d-glucosamine-specific lectin purified from Vibrio cholerae 01

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.     

Protective activity of glycolipid antigen against infection by Leptospira interrogans serovar canicola

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

Affinity purification and partial characterization of IgM-like immunoglobulins of African catfish, Clarias gariepinus (Burchell, 1822)

IgM like macroglobulin from bovine serum albumin (BSA)-immunized African catfish C. gariepinus was purified by affinity chromatography and partially characterized. The molecular weight of this macroglobulin was 840 kDa, as estimated by gel filtration chromatography. Purified macroglobulin was analyzed using SDS-PAGE under reducing and non-reducing conditions. The molecular weight (MW) of heavy and light chain was 74.8 kDa and 27.2 kDa respectively, in presence of a reducing agent. In non-reducing SDS-PAGE, a single high MW band was observed representing tetrameric form.     

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.     

Some properties of monkey intestinal sucrase

A detergent solubilised sucrase from monkey small intestine has been purified 388-fold to gel electrophoretic homogeneity with an overall recovery of 36%. The molecular weight of the enzyme was 263 kDa by gel filtration. Electrophoresis in the presence of SDS indicates that the enzyme is a hetero-dimer. Mixed substrate inhibition studies and inhibition by PCMB and Tris suggest the presence of two catalytically active sites in the form of maltase and sucrase with isomaltase activity being common to both sites. Polyclonal antiserum against the purified enzyme showed a single continuous precipitin line with the purified antigen.     

Improved protection against lung colonization by Actinobacillus pleuropneumoniae ghosts: characterization of a genetically inactivated vaccine

Pigs immunized with Actinobacillus pleuropneumoniae ghosts or a formalin-inactivated bacterin were found to be protected against clinical disease in both vaccination groups, whereas colonization of the lungs with A. pleuropneumoniae was only prevented in ghost-vaccinated pigs. Bacterial ghosts are empty cell envelopes created by the expression of a cloned bacteriophage lysis gene and, unlike formalin-inactivated bacteria, suffer no denaturing steps during their production. This quality may lead to a superior presentation of surface antigens to the immune system. Analysis by SDS-PAGE and immunoblotting of the two vaccine preparations revealed different contents of antigenic proteins. In order to better understand the immunogenic properties of A. pleuropneumoniae ghosts and formalin-inactivated bacteria, we compared the serum antibody response induced in both treatment groups. Immune sera were tested on whole cell antigen or purified virulence factors including outer membrane protein preparations (OMPs), outer membrane lipoprotein OmlA1, transferrin binding proteins (TfbA1, TfbA7 and TfbB) and Apx toxins (ApxI, II and III). SDS-PAGE and immunoblots revealed no specific antibody response against the single virulence factors tested in any vaccinated animal. The two vaccination groups showed different recognition patterns of whole cell antigen and OMP-enriched preparations. A 100 kDa protein was recognized significantly stronger by ghost-vaccinated pigs than convalescent pigs. This unique antibody population induced by ghosts could play a determining role in the prevention of lung colonization. The same 100 kDa antigen was recognized by ghost-sera in homologous as well as heterologous serotype A. pleuropneumoniae protein preparations. Indications for a crossprotective potential in the ghost vaccine were supported by studies on rabbit hyperimmune sera.     

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon,Thermoplasma acidophilum

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.     

A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli

MANB36, a secrete endo-beta-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 degrees C. The enzyme activity of MANB36 is remarkably thermostable at 60 degrees C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.     

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.     

Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.