Genomic and cDNA Cloning,Expression, Purification,and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds

A 516-bp winged bean chymotrypsin-trypsin inhibitor (WbCTI) gene was amplified from genomic DNA and cDNA isolated from winged bean using a pair of degenerate primers designed on the basis of the amino acid sequences of WbCTI. The amplified PCR products were cloned and sequenced to confirm their authenticity. DNA sequence analysis of the genomic and cDNA clones of WbCTI revealed the same nucleotide sequence in the coding region and showed WbCTI to be an intron less gene. WbCTI was subcloned into pTrc99A and expressed in Escherichia coli to yield a recombinant protein (rWbCTI). rWbCTI was purified by a rapid and single step immunoaffinity chromatography method, with an overall yield of 1.1 mg/g of wet cells. The homogeneity of the purified protein was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed the presence of a single protein band. Functionally rWbCTI is indistinguishable from WbCTI, since both inhibit bovine trypsin and chymotrypsin in a 1:1 molar ratio. FPLC binding studies also confirmed that rWbCTI binds the proteases in a 1:1 molar ratio.     

红桂木凝集素的纯化与性质研究

红桂木（Ａｒｔｏｃａｒｐｕｓｌｉｎｇｎａｎｅｎｓｉｓ）、俗名胭脂,属桑科桂木属,为亚热带、热带植物．红桂木种子含丰富的红桂木凝集素（Ａｒｔｏｃａｒｐｕｓｌｉｎｇｎａｎｅｎｓｉｓｌｅｃｔｉｎ,ＡＬＬ）,但迄今国内外均未见关于它的报道．我们采用Ｇａｌ－Ｓ...     

Purification and Characterization of a Lectin from Crotalaria paulina Seeds

A lectin was purified from Crotalaria paulina seeds by ion-exchange and FPLC molecular exclusion chromatography. CrpL had an apparent molecular mass of 30 kDa, as determined by SDS-PAGE under non-reducing and reducing conditions. CrpL effectively agglutinated human and cow erythrocytes, and this activity was not affected by 20 mM EDTA, showing no dependence of divalent cations. Hemagglutination was inhibited by N-acetyl- D-galactosamine, D-galactose and was also inhibited by glycoproteins, fetuin and asialofetuin. The N-terminal amino acid sequence of CrpL was identical to those of other lectins from the genus Crotalaria, and amino acid composition showed high amounts of Asx and Glx, and was rich in Gly, Ala and Ser, as also reported for lectins from other Crotalaria species. CrpL inhibited the growth of Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. passiflorae, suggesting a role of this lectin in the defense of seeds against bacterial infections.     

Purification and Characterization of Lectin from Seeds of Delonix regia

A lectin from the crude extract of seeds of Delonix regia (DRL) has been purified by ammonium sulphate fractionation followed by specific adsorption on Sephadex G-50 column and subsequent displacement with 100 mM D-glucose. The purified lectin (yield 1.41 mg g^?1 dry seed) is a hetero-tetramer of 156 kD in size, consisting of four polypeptides (M_r of 32, 36, 42 and 46 kD) as detected on SDS-PAGE. It is a thermostable protein and remains active between pH 2.0–11.0. The lectin agglutinated erythrocytes of human and other primates. The hemagglutinating activity was not affected by cations and chelating agents. Of the 23 different sugars tested for specificity, maximum inhibition of the hemagglutination was shown by D-glucose. The immunological crossreactions of DRL with monospecific antibodies against SBA, Con A, PNA, DBA and PHA-E indicate that DRL is very closely related to Concanavalin A.     

白桂木凝集素的纯化与性质的研究

白桂木种子粉经抽提、３０～６０％饱和度硫酸铵沉淀和亲和层析,已获得纯化的白桂木凝集素（Ａｒｔｏｃａｒｐｕｓｈｙｐａｒｇｙｒｂｕｓｌｅｃｔｉｎ）。浓度梯度ＰＡＧＥ,显示基本是均一的蛋白质带,分析表明,它是由分子量为１５０００和１９０００两种亚基组成,Ｎ端为精氨酸和丙氨酸,ｐＩ９．１０、８．４５、８．１０,中性精含量约６．９％,氨基糖约０．４％,能凝集多种动物红细胞和人Ａ、Ｂ、Ｏ和ＡＢ血型红细胞,凝集活力受Ｇａ１ＮＡｃ、Ｇａｌ和棉子糖的抑制,对热较敏感,在ｐＨ４．５～９．５的范围内．ｐＨ的改变不影响它的血凝活力。     

Purification and Characterization of a Lectin from Amaranthus hypochondriacus var. Mexico Seeds

A lectin from Amaranthus hypochondriacus var. Mexico (AHML) was purified by affinity chromatography using asialofetuin-Sepharose 4B. AHML is specific for N-acetyl-d-galactosamine as are the other Amaranthus lectins. AHML has no carbohydrate moiety and requires no metal ion for the hemagglutination activity. The pI of AHML is 6.8. AHML has a native molecular mass of 45.0 kDa and is composed of homo-subunits having molecular masses of 36.8 kDa.     

旋扭山绿豆（Desmodiumintortum）凝集素的纯化与特性

旋扭山绿豆（Ｄｅｓｍｏｄｉｕｍｉｎｔｏｒｔｕｍ）凝集素的纯化与特性彭建宗,程双奇,陈兆平,莫熙穆（华南师范大学生物系,广州５１０６３１）关键词凝集素;旋扭山绿豆;亲和层析;糖蛋白豆科植物和根瘤菌之间的共生结瘤固氮关系早有报道。例如大豆只被Ｂ．仰。６。...     

短裙竹荪(Dityophora duplicata)凝集素纯化与生化性质

短裙竹荪子实体经生理盐水抽提、硫酸铵沉淀、DEAE Sepharose和SephadexG 10 0柱层析纯化得到短裙竹荪凝集素 (Dityophoraduplicata(Bosc)Fischerlectin) ,简称DDFL .DDFL经PAGE显示单一条带 ,SDS PAGE测得其亚基分子量为 2 2 3kD ,SephadexG 10 0凝胶过滤测得分子量为 4 5 3kD ,DDFL不含中性糖 ,IEF测得其等电点为 3 92 .该凝集素对供试的 4种血型人血和兔、小牛、鸭、鸡、鲫鱼以及青蛙血红细胞具有凝集作用 ,但不凝集鳖红细胞 .它还可以凝集小鼠脾脏淋巴细胞和小鼠S180 肉瘤细胞 ,对兔红细胞的凝集作用可被乳糖、棉子糖、半乳糖、α 甲基半乳糖、β 甲基半乳糖和N 乙酰半乳糖胺所抑制 .氨基酸组成分析表明 ,DDFL含有 17种氨基酸 ,其中天冬氨酸、丝氨酸、苯丙氨酸和丙氨酸含量较高 .经测定 ,其N末端为甘氨酸 .DDFL对热、酸和碱具有一定的稳定性 ,经 6 0℃处理 10min ,可保持较高的活性 ,在pH 4 0～ 9 0范围内较稳定 ,其凝血活性依赖于Mg2 + 和Ca2 + 二价阳离子 ,Mn2 + 和Zn2 + 则无影响 .DDFL对小鼠腹腔注射的半致死量为 70 6 3mg kg .     

Purification and Characterization of Protease Inhibitor from Rice Bean (Vigna umbellata T.) Seeds

A Protease inhibitor from seeds of rice bean has been purified to apparent homogeneity as judged by native-PAGE with about 29% recovery using ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration through Sephadex G-100. The purified preparation with molecular weight of 16.8 kD was found to be a monomer as revealed by SDS-PAGE under reducing and non-reducing conditions. The purified inhibitor in solution was stable upto 40°C. However, it lost its activity gradually and was completely inactive when heated at 100°C for 2 h and 125°C for 1 h. Heating at 125°C changed the conformation of the inhibitor as was evident from the altered UV spectrum compared to that of native. It had two pH optima at pH 6.0 and at 10.0 and was stable over a wide range of pH (pH 3.0 to 10.0). It lost its activity on exposure to 2-mercaptoethanol, indicating the role of S-S linkages in maintaining the three dimensional structure of the protein inhibitor. The inhibitor was completely inactive towards papain, while it inhibited pepsin only slightly. Trypsin and chymotrypsin were inhibited upto the extent of 60% and 30%, respectively. Trypsin inhibition was of non-competitive type with dissociation constant for the enzyme-inhibitor complex in the region of 2.07 mg ml^-1. The rice bean inhibitor appears to be of Bowman-Birk type as it has molecular weight lower than that generally observed for Kunitz type inhibitors and seems to be double headed - a characteristic specific of Bowman - Birk type inhibitor.     

Single Step Purification,Characterization and N-Terminal Sequences of a Mannose Specific Lectin from Mulberry Seeds

A mannose/glucose specific lectin have been isolated and purified from mulberry seeds by affinity chromatography on ConA Sepharose. The lectin is monomer in nature as judged by SDS-PAGE and its MW was estimated to be 22,000. The lectin is glycoprotein with neutral sugar content of 28.57%, and mannose and glucose were identified as carbohydrate. The lectin agglutinated rat red blood cells and in a hapten inhibition test, D-mannose and D-glucose was found to be inhibitor. The lectin also exhibited cytotoxic effect in brine shrimp lethality bioassay. The N-terminal sequences of the lectin upto 45-residues except the positions of 21, 39, 42 and 44 were identified. Sequence homology of the lectin is also discussed.     

Purification and Characterization of Lectin from Lathyrus sativus

Lectin has been isolated and purified from Lathyrus sativus using ammonium sulphate precipitation followed by affinity chromatography. The molecular weight as determined by HPLC was found to be 42kD. The lectin is a tetramer, consisting of two types of subunits of which the heavier subunit consists of 2 polypeptides of mol wt of about 21 kD and 16 kD while the smaller subunits consists of two polypeptides of about 5kD as revealed by SDS-PAGE. The most potent sugar inhibitor of the Lathyrus lectin was found to be α-methyl D-mannoside. The N-terminal amino acid sequence was similar to that of pea lectin sequence.     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

Purification and Characterization of Cotton Lectin

Defatted seeds of wilt-disease resistant were extracted overnight with PBS at 4 ℃. After centrifugation. 90% saturated (NH4)2SO4 was added to the supernatant. The precipitates were dialysed against H2O, then lyophilized. The purified lectin was obtained by DEAE-cellulose ion-exchange chromatography, Sephadex G-100 filtration, and Sepharose 4B-Hog thyroglohulin affinity chromatography. The activity of the lectin was tested with fresh rabbit erythrocyte in each step of the procedure, and the active part was collected. This sample demonstrated single band on PAGE, SDS-PAGE and HPLC. The lectin was a glycoprotein. It contained 1.5% of neutral saccharide and its molecular weight was 63000 determined by Sephadex G-100 filtration. The N-terminal amino acid of the lectin was Val. The lectin showed no specific agglutination with any type of human erythrocytes. The hemagglutinaition activity could be inhibited by galactose and hog thyroglobilin, and depended on Ca2+, Mn2+, especially on Ca2+, not Mg2+. Its biological activity lost at 65 ℃ for S min. This lectin is used as a mitogen for human peripheral blood lymphocytes.     

Purification and Characterization of a Magnesium Ion Requiring N-Acetyl-D-glucosamine Specific Lectin from Seeds of Quercus ilex L.

A new magnesium ion requiring N-acetyl-D-glucosamine specific lectin QIL was purified to electrophoretic homogeneity from seeds of Quercus ilex L. through successive steps of (i) lectin extraction, (ii) ammonium sulphate (30–50%) fractionation, (iii) diethylaminoethyl (DEAE)-cellulose chromatography, (iv) carboxymethyl (CM)-cellulose chromatography, and (v) Sephadex G-75 chromatography. The lectin, having specific activity of 25,600 hemagglutination units (HAU)/mg of protein, was found to be a monomeric protein with a native molecular weight of 13.2 kDa. N-Acetyl-D-glucosamine was found to exhibit most potent inhibitory action on the lectin activity among all the sugars tested. The lectin was also found to exhibit specificity for human blood groups A, B, and AB. It was converted to the corresponding apo-lectin by ethylenediaminetetraacetic acid (EDTA) treatment followed by buffer dialysis. The apo-lectin exhibited a specific and characteristic requirement for magnesium ions for the expression of its activity.     

Purification,Characterization, and Antiproliferative Activity of a Single-Chain Lectin from Vicia palaestina (Fabaceae) Seeds

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity.     

三齿草藤(Vicia bungei Ohwi)凝集素的亲和层析纯化及其部分性质的研究

用Sephadex G-100或猪甲状腺球蛋白-对氨基苯砜乙基-交联琼脂作亲和吸附剂,均可从三齿草藤(Vicia Bungei Ohwi)的种子中分离纯化出三齿草藤凝集素。该凝集素经连续或不连续系统聚丙烯酰胺凝胶电泳均显示出单一蛋白带;糖蛋白染色法证实为糖蛋白;SDS-聚丙烯酰胺凝胶电泳测定其分子量为24,600,凝集素浓度为1.95微克/毫升时就能凝集兔红细胞;但对人ABO型血细胞不发生凝集作用;其对兔红细胞的凝集作用可被D-Man、D-GlcNA和D-GIC所抑制;它也是一种促有丝分裂原。     

香灰菌凝集素的纯化及其部分性质

香灰菌菌丝体经磷酸缓冲液抽提、20%-70%饱和浓度的硫酸铵沉淀、DEAE-Cellulose和SephadexG-100柱层析纯化得到香灰菌凝集素(Hypoxylonsp.lectin,简称HSL)。HSL经PAGE检测为单一蛋白条带,SDS-PAGE测得其亚基分子量为15.9kD。过碘酸-Schiff染色法表明HSL为一种糖蛋白,糖基的含量为15.5%,β-消去反应测得其糖和蛋白质的连接键为O-型糖肽键。HSL能凝集多种动物红细胞和人的红细胞,在所测试的红细胞中,对兔红细胞的凝集作用最强。HSL对热较敏感,经50°C处理10min,其凝集活性明显降低,其在碱性环境中较稳定,而在酸性环境中较不稳定。HSL的凝集活性受Al3+、Fe3+、Ca2+和Zn2+等阳离子的影响。对鼠红细胞的凝集作用可被半乳糖和乳糖所抑制。     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Ｓｅｐｈａｒｏｓｅ ６Ｂ亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素（ＢＶＬ）,其比活性比抽提液提高了１５９倍,活力回收率为４９．０％。ＢＶＬ分子量为８１０００,由两个相同的亚基组成。等电聚焦凝胶电注测得其等电点为４．９５。其紫外吸收高峰在２７６ｎｍ处。ＢＶＬ具一定的的热稳定性和酸碱稳定性。Ｎ－乙酰－Ｄ氨基半乳能强烈地抑制ＢＶＬ对兔红细胞的凝集作用。乳糖、半乳糖也有较强的抑制定性。Ｎ     

Purification and Characterization of the Millettia pachycarpa Lectin

A lectin with strong hemagglutinating and mitogenic activity was isolated from the seeds of Millettia pachycarpa Benth. by extraction, fraction with (NH4)2 804, ion-exchange chromatography on DEAE-Sepharose and followed by gel filtration on Sephadex G-100. The purified leetin showed a single protein band on PAGE and SDS-PAGE, and exhibited a molecular weight of 40 700 by gel filtration and subunit of 19 800 on SDS-PAGE. It contained 1.78% neutral saceharide and enriched Asp, Glu, Thr, Set, Leu and also contained 4 Trp per molecule. It agglutinated rabbit red cell at 0.48 μg/mL and A,B,O types of blood. The reaction could be inhibited by thyroglobulin, muein gastrie and ovomucin, but not by saceharide. The hemagglutination was strongly dependant on Ca2 +, but was not enhanced by Mg2 + ,Mn2 + ,Zn2 +; The lectin was a strong mitogen for human peripheral blood lymphoeytes, showing a transformation rate and mitotic index of 84.3 % and 7.8 %,respectively.     

洋紫荆凝集素的分离纯化和性质

利用酸化处理的Sepharose 6B 亲和柱从洋紫荆种子中分离纯化出了洋紫荆凝集素(BVL),其比活性比抽提液提高了159倍,活力回收率为49.0%。BVL分子量为81 000,由两个相同的亚基组成。等电聚焦凝胶电泳测得其等电点为4.95。其紫外吸收高峰在276 nm处。BVL具一定的热稳定性和酸碱稳定性。N-乙酰-D-氨基半乳糖能强烈地抑制BVL对兔红细胞的凝集作用,乳糖、半乳糖也有较强的抑制作用。