Purification and properties of chicken egg-white cobalamin-binding protein

A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities.     

重组尿激酶原的纯化和性质研究

CHO工程细胞11G持续表达的pro-UK分泌在细胞培养液的上清中,培养液上清经过微孔玻璃(MPG)吸附色谱,羧甲基阳离子交换色谱,高压液相凝胶色谱三步纯化,纯化倍数可达700倍以上,总回收率为46％．再经过Benzamidine-Sepharose 6B亲和层析去掉少量的双链尿激酶,得到纯化尿激酶原．终产物经SDS-PAGE银染分析,纯度达90％以上,分子量为52 ku,其比活性为51 220 U/mg．抗体中和、二异丙基氟磷酸(DFP)抑制等实验证明重组pro-UK的性质和天然pro-UK的性质相一致．     

Hydrophobic chromatographic purification of ethylene-enhanced chlorophyllase from Citrus unshiu fruits

Ethylene-enhanced chlorophyllase from Citrus unshiu fruits was purified to a homogeneous state after solubilization with sodium cholate, using acetone precipitation and hydrophobic chromatography. The enzyme adhered to phenyl Sepharose CL-4B in 3M KCl and was eluted with a linear gradient of Triton X-100 (0–0.5%). Its MW (SDS-PAGE) was 27 000. The enzyme behaved as a protein of MW 110 000 on Sephacryl S-200 gel filtration. The enzyme showed a specific activity of 0.069 μamol chlorophyllide a produced/min/ mg protein. This purification procedure is a rapid method for obtaining pure chlorophyllase.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

A simple high-yield procedure for isolation of human urinary kallikreins.

Two human urinary kallikreins (fractions A-1 and A-2) were purified to apparently homogeneous forms. The two kallikreins were separated by affinity chromatography using Trasylol (aprotinin) covalently bound to Sepharose. The kallikreins were eluted with a pH gradient (pH 9.5-3.0). Fraction A-1 was eluted between pH 6.2 and 4.2 and fraction A-2 was eluted between pH 4.2 and 3.1. Final purification was obtained by chromatography on Sephacryl SS-200. Antibodies prepared against fraction A-2 were also reactive with fraction A-1; thus it may be possible to measure both by radioimmunoassay using the same antibody preparation.     

Purification of soluble acetylcholinesterase from Japanese quail brain by affinity chromatography.

The purification of a soluble acetylcholinesterase from Japanese quail brain using affinity chromatography on concanavalin A-Sepharose and edrophonium-Sepharose is described. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose. Acetylcholinesterase was purified 10,416-fold with a specific activity of 2500 U/mg protein. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol gave only one band with a molecular weight of 62.5 kDa. The molecular weight of the purified acetylcholinesterase was estimated to be 245.5 kDa by gel chromatography on Sephacryl S-200 under nondenaturing conditions. Based on the molecular weight obtained by both SDS-PAGE and gel filtration the purified acetylcholinesterase was assumed to be a tetrameric form.     

Isolation,Purification and Some Properties of Invertase from Leaves of Mandarin Orange

Highly active acid invertase was found in the young leaf extract of mandarin orange Citrus reticulata Blanco). The invertase was isolated and purified from the young leaf extract of mandarin orange through the procedures of ammonium sulphate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. 6.4% of the invertase activity was recovered. Invertase was 179.2-folds purified. The purified invertase preparation was homogeneous as shown in polyacrylamide gel electrophoresis and Sephacryl S-200 molecular sieve chromatography. The molecular weight of the native invertase determined by gel filtration was 80 kD. The invertase consists of two identical subunits with apparent equal subunit weight of 40 kD as determined on SDS-PAGE. The invertase followed typical Michaelis-Menten Kinetics with apparent Km Of 1. 6 × 10-2 mol/L for sucrose. Vmax of the invertase was 100 mg reducing sugar · mg-1 protein · h-1 The optimum pH was 5.0 (stable from 4.5—5.5). The optimum temperature was 55℃.     

Effective method for purification of lysozyme from human urine

Lysozyme in the urine of a hemodialysis patient was purified in two steps: DEAE Sephadex chromatography followed by Sephacryl chromatography. The Sephacryl S-100 column chromatographed fraction showing lytic activity was proven to give one band on SDS-PAGE and to have a molecular mass of 14 500, in agreement with that of lysozyme. The N-terminal amino acid sequence of this purified protein was identical to that of lysozyme. These results indicate that the protein purified was indeed lysozyme. The specific affinity of lysozyme for Sephacryl S-100 may explain the greater purity of the same protein isolated by this method.     

Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor

We have identified a tyrosine kinase activity present in tumors which were raised in rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This kinase phosphorylates tyrosine on the heavy chain of IgG from tumor-bearing rabbit (TBR) sera specific for the src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we have purified this kinase over 7200-fold. The purification procedure involves detergent extraction of tumors followed by sequential column chromatography on hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, an affinity column prepared from TBR-sera, and Sephacryl S-200. The IgG kinase activity behaves as a molecule of apparent Mr = 54,000 on Sephacryl S-200 molecular sieve chromatography. Analysis of the Sephacryl fractions by SDS-PAGE indicates that a major Coomassie blue-stained band with an apparent Mr = 54,000 (p54), co-elutes with the peak of kinase activity. From 600 g of tumors, approximately 200 micrograms of p54 are obtained. We have four types of evidence which show that p54 is related to pp60src. 1) Purified p54 is capable of undergoing endogenous phosphorylation in the presence of [gamma-32P]ATP producing a 32P-labeled pp54 polypeptide which is specifically immunoprecipitated by TBR-sera and contains only phosphotyrosine. 2) Purified p54 competes with 32P-labeled pp60src for binding to TBR-IgG, indicating a degree of purification over starting material which agrees very well with the results obtained by the IgG kinase assay. 3) V8 protease digestion of pp60src and p54 suggests that they share a common 26,000 fragment. 4) Antibodies to partially purified p54 specifically precipitate pp60src from Rous sarcoma virus-transformed chicken cells.     

Purification and some properties of an extracellular ribonuclease with antiviral activity against tobacco mosaic virus from <Emphasis Type="Italic">Bacillus cereus</Emphasis>

A new ribonuclease (RNase) with tobacco mosaic virus inhibition was isolated and purified from Bacillus cereus ZH14 through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography of DEAE-Sephadex A-50 column, and gel chromatography of Sephacryl S-200HR column. The enzyme was purified approximately 134-fold with a recovery of 9.2%. The RNase had an MW of 75.6 kDa in SDS-PAGE, which differed from RNases reported previously. The inhibitory activity of the RNase in the purification process against tobacco mosaic virus was tested, and the percentage inhibition of the purified RNase (48 U/ml) reached 90%. The protein could tolerate 90°C and pH 4.0.     

A 60-kDa phosphorylated protein from fetal human bone

A phosphorylated protein was isolated and purified from fetal human bone. Fetal and adult human bones were decalcified with EDTA, and the extract from the fetal bone was fractionated using Q-Sepharose anion exchange chromatography. The fraction containing Ser(P) was purified by Sephacryl S-200 molecular sieving and C4 reverse-phase HPLC. The purified protein had a molecular weight of 60000 on SDS-PAGE, where the protein was stained with Rhodamine-B. The amino acid composition of this protein was different from any other reported phosphorylated proteins in human bone. However, this phosphorylated protein was difficult to detect in the adult bone extract on SDS-PAGE.     

纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

刺芹侧耳芳基醇氧化酶的分离纯化及其酶学性质

分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70kDa和4.2。以藜芦醇为底物,该酶最适反应pH为6.0,最适反应温度为70℃,金属离子Zn²⁺、Fe²⁺和Cu²⁺对芳基醇氧化酶的活性抑制作用明显,K_m和V_max分别为0.921mmol/L和80U/mg。     

囊尾蚴膜联蛋白32在大肠杆菌中的表达及纯化

在已获得的囊尾蚴膜联蛋白 (Annexin) 32cDNA的基础上 ,以PCR的方法在cDNA两端加上酶切位点 ,插入原核表达载体pJLA 5 0 3,热诱导表达后 ,外源蛋白大部分以可溶形式表达 ,表达量占菌体总蛋白的 35 %。经(NH₄)₂SO₄分级沉淀、DEAE阴离子交换层析及Sephacry1S 2 0 0凝胶过滤层析得到电泳单一条带 ,Westernblot及抗凝血实验证明表达产物是有生物活性的     

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.     

Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3

The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3.     

Subunit composition and Ca(2+)-ATPase activity of the vacuolar ATPase from barley roots.

The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.     

Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic <Emphasis Type="Italic">Nesterenkonia</Emphasis> sp. strain F

A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase activity was stimulated by Ca²⁺ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Simultaneous isolation of prolactin and growth hormone from "discarded acid pellet" obtained from buffalo pituitaries

An acid pellet, obtained as a side fraction from a conventional gonadotropin purification pathway, has been found to contain the bulk of the pituitary lactogenic hormones (growth hormone or GH and prolactin or PRL). This discarded side fraction has been utilized to obtain buffalo lactogenic hormones (buGH and buPRL), simultaneously, and in bulk. The immunoreactivities of the purified semi-pure buffalo GH and PRL (APECS and APP-I, respectively) preparations were compared by direct binding ELISA with semi-pure standard buGH and PRL (ECS and EP-I, respectively) and were found to be as pure as standard semi-pure buGH and buPRL. When checked by direct binding ELISA using buGH and buPRL antisera, it was observed that APECS and APP-I were not cross-immunoreactive. SDS-PAGE and western blot analysis of APECS and APP-I showed major bands located at the same positions as in the case of standard semi-pure preparations (20 kDa for APECS and 23 kDa for APP-I). The semi-purified buGH and buPRL (APECS and APP-I) were converted to a highly purified preparation by chromatographing them via Sephacryl S-200 gel-filtration chromatography.