Isolation,purification, and N-terminal partial sequence of an antitumor peptide from the venom of the Chinese scorpion Buthus martensii Karsch

An antitumor peptide (ANTP) was isolated and purified from the venom of the Chinese scorpion Buthus martensii Karsch. The purification procedure included gel filtration on Sephadex G-50 and Superdex 30 high resolution chromatography, Phenyl Sepharose 6 Fast Flow chromatography, and SP-Sepharose Fast Flow chromatography. Its homogeneity was demonstrated by size exclusion HPLC on TSK G2000 SW. The isoelectric point is more than 10 by pH 3-10 range isoelectric focusing. ANTP has a relative molecular mass of 6280, calculated from the measurement of 16.5% SDS-PAGE. The pharmacological tests showed that ANTP has antitumoral effects in the mouse S-180 fibrosarcoma model and Ehrlich ascites tumor model. Amino acid analysis suggested the ANTP is rich in glycine and does not have histidine and threonine. The sequence of the first 25 N-terminal residues is as follows: Val-Arg-Asp-Gly-Tyr-Ile-Ala-Asp-Asp-Lys-Asn-Cys-Ala-Tyr-Phe-Cys-Gly-Arg-Asn-Ala-Tyr-Cys-Asp-Asp-Glu.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

Purification of Bovine α-Lactalbumin by Immobilized Metal Ion Affinity Chromatography

ABSTRACT

The milk protein α-lactalbumin was isolated from bovine whey protein concentrate solution by immobilized metal ion affinity chromatography (MAC) using Cu(II)-Chelating Sepharose Fast Flow. Stepwise pH (5.5–3.8) changes in sodium acetate buffer were used to elute the protein selectively, at which time it was concentrated and reapplied to an uncharged Chelating Sepharose Fast Flow column to remove the contaminating Cu(II) ions. A purity of 90% and recovery of 80% was achieved. The described method appears to be suitable for isolation of a-lactalbumin in a form adequate for milk formula engineering.     

Purification Process for the Preparation and Characterizations of Hen Egg White Ovalbumin,Lysozyme, Ovotransferrin,and Ovomucoid

Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

A Novel Phospholipase C Inhibitor,S-PLI Produced by Streptomyces Sp. Strain No. A-6288

Abstract

S-PLI, an inhibitor of phospholipase C (PLC) produced by Strepromyces sp. strain No. 6288, was purified from the culture filtrate by salting-out with solid ammonium sulfate, column chromatography on CM-cellulose and gel filtration on Sephadex G-75. The molecular weight of S-PLI was estimated to be 65,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was found to be a glycoprotein with a composition of 609 amino acids and 19 glucose residues having an isoelectric point at 7.8. S-PLI was stable from pH 3 to 10 at 37°C and up to 40° at pH 6.0. The inhibitory activity showed pH-and temperature-dependence with a maximum around pH 7.0 at 50°C. S-PLI inhibited phospholipase C in a competitive manner (K_i value; 9.5 × 10-⁶ mM), but did not inhibit S-Hemolysin, phospholipase A₂, phospholipase B, phospholipase D and phosphatases. S-PLI is the first reported example of a glycoproteinaceous inhibitor of microbial origin which is able to specifically inhibit phospholipase C.     

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

Abstract

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400?SEC, we obtained highly pure, i.e.,?>?90%, rHBsAg virus-like particles (VLP).     

<Emphasis Type="Italic">Aspergillus niger</Emphasis> lipase: gene cloning,over-expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> and in vitro refolding

From the N-terminal amino acid sequence of the lipase from Aspergillus niger F044, a potential homologous gene A84689 to the lipanl (the gene encoding the lipase from Aspergillus niger F044) was identified. A pair of primers were designed according to the nucleotide sequence of A84689, and the lipanl was cloned by PCR. Nucleotide sequencing revealed that the lipanl has an ORF of 1,044 bp, containing three introns. The deduced amino acid sequence corresponds to 297 amino acid residues. The cloned cDNA fragment encoding the mature lipase from Aspergillus niger F044 was over-expressed in Escherichia coli BL21(De3) and the recombinant protein was refolded in vitro by dilution followed by DEAE Sepharose Fast Flow chromatography.     

Pilot-scale fermentation, purification, and characterization of recombinant human Oncostatin M in Pichia pastoris

Oncostatin M (OSM) is a multifunctional cellular regulator that belongs to the IL-6 subfamily and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. In order to achieve the higher level yield of recombinant human Oncostatin M (rhOSM), we determined the optimal pH condition of rhOSM expressed in the methylotrophic yeast Pichia pastoris X-33 and carried out the fermentation culture of rhOSM in 80 L fermentor in a fed-batch mode. SDS–PAGE and Western blotting assays demonstrated that rhOSM was successfully expressed and secreted into the culture medium with an apparent molecular weight of 28 kDa. N-terminals were correctly processed through amino-terminal sequencing. The maximum yield of rhOSM was 280 mg/L. rhOSM was purified by phenyl Sepharose hydrophobic interaction chromatography and SP Sepharose Fast Flow cation exchange chromatography, which resulted in a final yield of purified rhOSM of 6.94 g with a recovery of 62% and a purity of 95%. The purified rhOSM had a specific growth inhibition activity of 6.26 × 10⁴ RU/μg, which was commensurate with typical values (6.2 × 10⁴ RU/μg) obtained with standard hOSM.     

Isolation and Structural Analysis of an Acidic Polysaccharide from Astragalus membranaceus （Fisch.） Bunge

A new water-soluble hetero-polysaccharlde, APSID3, was obtained from a hot-water extract of the roots of Astragalus membranaceus （Flsch.） Bunge by DEAE-Sepharose Fast Flow and Sephacryl S-300 chromatography. The molecular weight of APSID3 was estimated to be 5.79 × 10^5 Da. Based on a sugar composlUon analysis, methylatlon analysis, partial hydrolysis and 13C nuclear magnetic resonance experimentation, It was concluded that the minimal repeat unit of APSID3 was composed of one terminal arablnose, one 1,5-1Inked arabinose, one 1,3-1Inked rhamnose, one 1,3,4-1Inked rhamnose, five 1,4-1Inked methyl galacturonates and six 1,4-1inked methyl glucuronates.     

枯草芽孢杆菌中性β-甘露聚糖酶的纯化及性质研究

经硫酸铵分级沉淀、超滤浓缩、阴离子交换层析和凝胶过滤层析,由枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）ＢＭ９６０２培养滤液得到了常规凝胶电泳一条带的中性β－甘露聚糖酶．该酶具有与其它已知同类酶相类似的性质,但用ＳＤＳ－ＰＡＧＥ测得该酶分子量为３７ｋＤ;用聚丙烯酰胺等电聚焦电泳测得等电点ｐＩ为４．９．     

Isolation and antioxidant property of the extracellular polysaccharide from <Emphasis Type="Italic">Rhodella reticulata</Emphasis>

The extracellular polysaccharide from Rhodella reticulata was separated from the culture medium followed by concentration and ethanol precipitation, and purified by anion exchange chromatography on DEAE-Sepharose Fast Flow. This study compared the free radical-scavenging property and antioxidant activity with various treatments of crude extracellular polysaccharides of R. reticulata. The results showed that both the crude extracellular polysaccharide and deproteinized crude extracellular polysaccharide gave evidence of the free radical scavenging and antioxidant activity in a dose-dependent manner. The crude extracellular polysaccharide exhibited higher free radical scavenging capacity and better antioxidant activity than the various treatments of crude extracellular polysaccharide samples. The superoxide anion radical scavenging ability of various samples was significantly higher compared to standard antioxidant (α-tocopherol). These results indicate that the extracellular polysaccharide of R. reticulata is a potent natural antioxidant.     

大肠杆菌冷休克蛋白CspC的分离纯化及部分性质测定

经Sepharose Q Fast Flow阴离子交换层析和Superdex 30凝胶过滤层析,从大肠杆菌（Escherichia coli）细胞内分离纯化了一种小分子蛋白质，SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯度鉴定为单一条带，经质谱分析、N端测序、同源序列比较，确定该蛋白质为大肠杆菌冷休克蛋白CspC.在此基础上，用圆二色光谱测定了其二级结构含量，初步探索了其热稳定性及与单链DNA结合后的构象变化.     

PURIFICATION AND CHARACTERIZATION OF THE 20S PROTEASOME FROM THEALGA CHARA CORALLINA (CHAROPHYCEAE)1

We purified the 20S proteasome from the alga Chara corallina Willd with DEAE–ion‐exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme bynondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two‐dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin‐like, trypsin‐like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin‐like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin‐like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin‐like activity was inhibited by N‐ethylmaleimide, p‐chloromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin‐like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.     

Cloning, Overexpression, Refolding, and Purification of the Nonspecific Phospholipase C fromBacillus cereus

Bacillus cereussecretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester.B. cereusPLC has been overexpressed with its signal sequence inEscherichia coliusing a T7 expression system. The expressed enzyme formed intracellular inclusion bodies which were solubilized in the presence of 8 urea. Renaturation was initiated by gradual removal of urea and addition of zinc ions. The signal peptide was specifically cleaved by a protease, clostripain, added when the urea concentration was 1.5 . Factors that led to protein reaggregation included rapid removal of urea, use of Tris instead of barbital buffer, and presence of the signal peptide when the urea concentration was below 1.5 . The folded protein was purified by Q-Sepharose Fast Flow chromatography to yield a preparation >99% pure. The final yield of active enzyme was 30–40 mg per liter of culture. The recombinant PLC exhibited biochemical and kinetic properties identical to those of extracellularly produced PLC fromB. cereus.Site-specific mutagenesis of Asn-134 was carried out as a test of the general effectiveness of the refolding procedure.     

Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775)

A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forskål, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 0.125 mg/ml. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residue NH₂-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search in protein databases (BLAST) revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. We propose the name Scygonadin (from the gonad of S. serrata) for this antibacterial protein.     

One-step purification of xylanase from Melanocarpus albomyces and ethylene glycol as a novel soluble additive for enhancing its thermal stability

Hydrophobic interaction chromatography with Phenyl Sepharose 6 Fast Flow resulted in 7-fold purification of xylanaseactivity from Melanocarpus albomyces with over 60% recovery of theactivity. The purified preparation consisted of two major isoenzymes out of seven present in the organism. Ethyleneglycol, used as the eluent, enhanced the thermal stability of the xylanase activity from a half-life of 2.2 h at 55 °C to almost 40 h.     

Purification and physicochemical properties of different polysaccharide fractions from the water extract of Boschniakia rossica and their effect on macrophages activation

Today more and more attentions had been attracted by many nutritionists and pharmacologists on polysaccharides from natural plants or animals due to their significant biological activities. In this research three polysaccharides (BRR-W1, BRR-WA1 and BRR-WA2) were isolated and purified from the water extract of Boschniakia rossica by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Chemical and physical characteristics of three polysaccharides were investigated by a combination of chemical and instrumental analysis methods. The assays of their effect on macrophages activation were also investigated in vitro, including phagocytosis of macrophages, detections for NO production and TNF-α secretion. The results indicated that the effect of polysaccharides on macrophages activation was influenced by their respective physicochemical properties.     

Purification and Properties of Cytosolic Copper,Zinc Superoxide Dismutase from Watermelon (Citrullus vulgaris Schrad.) Cotyledons

Cytosolic copperzinc-superoxide dismutase (CuZn-SOD I; EC 1.15.1.1) was purified to homogeneity from watermelon (Citrullus vulgaris Schrad.) cotyledons. The stepwise purification procedure consisted of acetone precipitation, batch anion-exchange chromatography, anion-exchange Fast Protein Liquid Chromatography, gel-filtration column chromatography, and affinity chromatography on concanavalin A-Sepharose. CuZn-SOD I was purified 310-fold with a yield of 12.6 micrograms enzyme per gram cotyledons, and had a specific activity of 3, 540 units per milligram protein. The relative molecular mass for cytosolic CuZn-SOD was 34000, and it was composed by two equal subunits of 16.3 kDa. CuZn-SOD I did not contain neutral carbohydrates in its molecule, and its ultraviolet and visible absorption spectra showed two absorption maxima at 254 nm and 580 nm. Metal analysis showed that the enzyme contained 1 gram-atom Cu and 1 gram-atom Zn per mole dimer. Cytosolic CuZn-SOD was recognized by the antibody against peroxisomal CuZn-SOD from watermelon cotyledons, and its enzymatic activity was inhibited by this antibody. By IEF (pH 4.2–4.9), using a new method for vertical slab gels set up in our laboratory, purified cytosolic CuZn-SOD was resolved into two equal isoforms with isoelectric points of 4.63 and 4.66.