魔竽葡苷聚糖凝胶为亲和层析载体与Sepharose 4B的比较研究——Ⅰ.KGM金属螯和层析分离纯化猪血SOD

将KGM凝胶和Sepharose 4B在同样条件下活化偶联,制成Cu~(2 )金属螫合亲和胶,亲和纯化猪血SOD,并对这两种亲和胶的层析效果和性能进行了比较。KGM金属螫合胶对猪血SOD吸附量、纯化倍数、纯化SOD的比活力和回收率分别为53000U/ml胶、19倍、12000U/mg蛋白和94.6％,而Sepharose 4B亲和胶对SOD 的吸附量、纯化倍数、纯化SOD的比活力和回收率分别为79920U/ml胶、11倍、10125U/mg蛋白和95.4％。两种亲和胶所纯化的SOD经聚丙烯酰胺凝胶电泳(PAGE)、活性染色及SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)证明其均为电泳纯。KGM金属螯合胶使用六次后,其对SOD吸附量、去Cu量及SOD的回收率均无明显影响。     

魔竽葡苷聚糖凝胶为亲和导析载体与Sepharose 4B的比较研究

将KGM凝胶和Sepharose 4B在同样条件下活化偶联,制成Cu2 金属螯合亲和胶,亲和纯化猪血SOD,并对这两种亲和胶的层析效果帮性能进行了比较,KGM金属螯合胶对猪血SOD吸附量,纯化倍数,纯化SOD的比活力和回收率分别为53000U／ml胶,19倍,12000U/mg蛋白和94．6％,而epharose 4B亲和胶对SOD的吸附量,纯化倍数,纯化SOD的比活力和回收率分别为7992U／ml胶,11倍,10125U/mg蛋白和95．4％,两种亲和胶所纯化的SOD经聚丙烯酰胺凝胶电泳（PAGE）,活性染色及SDS聚丙烯酰胺凝胶电泳（SDS－PAGE）证明其均为电泳纯,KGM金属螯合胶使用六次后,其对SOD吸附量,去Cu量及SOD的回收率均无明显影响。     

一种新的植物低分子量ATPase的纯化及性质研究

采用丙酮粉技术,DEAE-Sephadex A50及FPLC离子交换层析技术从玉米花粉胞质中分离纯化了一种具有ATPase活性和GTPase活性的低分子量可溶性蛋白,纯化倍数为105倍.用SDS-PAGE、二维电泳及薄层扫描技术分析了分离样品的纯度.亚基分子量约为38ku,非变性PAGE测定的全酶分子量为76ku,表明该酶分子是由两个相同亚基组成的二聚体蛋白.等电聚焦电泳测定其等电点为5.6,是酸性蛋白质.用抗牛脑dynamin或kinesin的抗体进行Western-blotting,结果表明该酶蛋白与它们无免疫交叉反应.药理学研究表明:38ku蛋白对Na₃VO₄及NEM均非常敏感.     

眼镜蛇毒细胞毒素-4N的分离纯化及其对HSC-T6细胞的作用

为了得到高纯度眼镜蛇毒细胞毒素-4N,探索眼镜蛇毒中细胞毒素-4N(cytototxin-4N,CTX-4N)对大鼠肝星状细胞(hepatic stellate cells,HSC-T6)的增殖抑制作用。本研究采用DEAE-Sepharose CL-6B阴离子交换柱、Spehadex G-50凝胶层析柱、Macro-prep High S阳离子交换柱结合的方法对眼镜蛇毒蛋白进行分离纯化。在每一步分离纯化过程中,采用CCK-8法检测各蛋白峰组分对HSC-T6细胞的增殖抑制作用活性,收集增殖抑制作用最强的CTX-4N峰。经SDS-PAGE电泳鉴定蛋白纯度,Nano-LC-ESI-MS/MS质谱方法鉴定其组分,Cell Counting Kit-8(CCK-8)试剂检测CTX-4N对肝星状细胞(HSC-T6)的增殖抑制作用,从而确定其药理作用。经DEAE-Sepharose CL-6B阴离子交换柱、Spehadex G-50凝胶层析柱、Macro-prep High S阳离子交换柱分离纯化后得到一个电泳纯度的蛋白,蛋白质谱鉴定为CTX-4N,分子量约为9.605 k D。不同浓度的CTX-4N作用HSC-T6细胞24 h后,随着其浓度的增大对HSC-T6细胞增殖抑制作用越强,呈剂量-效应关系,IC_(50)为(12.836±0.045)μg/m L。因此,本研究建立一种眼镜蛇毒细胞毒素-4N的分离纯化方法,并确定了其对HSC-T6细胞的增殖抑制的药理作用随浓度的增加而增强,为进一步研究其药理作用提供一定的理论依据。     

重组人绒毛膜促性腺激素嵌合肽12的聚丙烯酰胺凝胶电泳制备

利用板状聚丙烯酰胺凝胶电泳 (PAGE)方法 ,建立了一步分离纯化基因工程表达蛋白质的新技术。在构建的人绒毛膜促性腺激素嵌合肽 (CP1 2 )表达率约为 1 %的情况下 ,借助分析制备两用型板状PAGE装置 ,通过下行和上行两次电泳以及用透析膜隔离形成收集槽这两步改进 ,从包涵体抽提液中一步纯化了目的表达产物。结果表明 ,一次上样总蛋白质量为 3～ 4mg的PAGE ,可获得 5 0～ 1 0 0 μgCP1 2蛋白 ,其相对均一性达到 95 %。研究提示此改良的制备性PAGE新技术 ,是一种分离纯化低分子量目的蛋白质的好方法     

毕赤酵母表达重组人白细胞介素11的纯化与鉴定

报道了毕赤酵母表达人白细胞介素11的下游工艺研究,并对其产物进行了分析鉴定。所用工艺流程为离心收集上清、超滤浓缩脱盐、离子交换层析、疏水层析、凝胶过滤。所得产物经SDSPAGE电泳、RPHPLC分析、N端和C端序列分析、质谱、等电点分析和生物学活性分析,结果表明：产品纯度大于97%,结构和性质与E.coli融合表达的Neumega完全一致。     

芜菁花叶病毒（广州油菜株）生化性质的研究

我们对芜菁花叶病毒广州油菜株(TuMV—G.Z)进行了生化性质的研究。结果表明,纯化的病毒外壳蛋白经10％SDS-聚丙烯胺酰胶电泳出,现一个组份,其分子量为1.4×10~4d;氨基酸组份分析表明,TuMV—G.Z外壳蛋白亚基由约107个氨基酸残基组成,其中包括1个色氨酸和5个酩氨酸;DNS—C1末端法测其N端,结果显示为苏氨酸,且其氨基是游离的。     

Erwinia herbicola冰核活性蛋白的分离、电泳分析鉴定

对Erwinia herbicola(A25)菌株的冰核活性蛋白的分离纯化及电泳进行研究。主要方法①按双温培养的方法,获得了较高的生物量积累和强冰核活性的诱导表达。②实验采用渗透压冲击法破碎细菌细胞,破碎率达98．67％。③通过差速离心法获取不同冰核活性蛋白组分,测定各组分的冰核活性和SDS－PAGE电泳图谱分析。建立了冰核活性因子的高冰核活性与离心力之间的关系;利用SDS－PAGE还建立了具冰核活性蛋白的分子量的大小与冰核活性蛋白组分之间的关系。证实了具高冰核活性蛋白质最小结构单位约为26．0kD。     

两种果子狸血清IgG纯化方法 的比较

目的 探讨一种具有简便快捷、高纯度、活性好的果子狸血清IgG纯化方法。方法 比较Hitrap Protein A亲和层析和PAGE电泳两种纯化法对果子狸血清IgG的纯化,用PAGE还原电泳和Western-Blot法对IgG作纯度鉴定。结果 对Hitrap Protein A纯化的果子狸血清IgG,其活性虽好,但纯度不高;而非还原PAGE电泳所纯化的果子狸血清IgG不但纯度高（〉95%）、并具有较强的免疫活性。结论 非还原PAGE电泳法纯化果子狸血清IgG,是一种具有高纯度、免疫活性强的纯化方法 。     

黑木耳漆酶纯化的研究

目的:研究黑木耳(Auricularia auricula)“黑29”的漆酶纯化,为进一步酶学性质的开展、酶应用和酶基因克隆提供理论基础。方法:采用硫酸铵分级沉淀和柱层析技术分离蛋白,通过PAGE和SDS-PAGE电泳检测蛋白。结果:SDS-PAGE电泳检测发现粗酶液含三种漆酶,分子量大小分别为LacA(60kD)、LacB(34kD)、LacC(19kD);纯化后获得纯化的单一漆酶LacA、LacC组分。结论:得漆酶两个单一组分,为进一步漆酶研究奠定基础。     

近江牡蛎铜锌超氧化物歧化酶的纯化及部分性质研究

经６５℃加热,硫酸铵分级沉淀,ＳｅｐｈａｄｅｘＧ－１００凝胶过滤和ＤＥ－５２柱层析,从近江牡蛎（ＯｓｔｒｅａｒｉｖｕｌａｒｉｓＧｏｕｌｄ）软体部分提纯了铜锌超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）．对其理化性质鉴定表明,用此法纯化的酶纯度均一．该酶系由两个相同亚基组成的二聚体,分子量２７．９ｋＤ．该酶的紫外吸收峰在２７２．５ｎｍ,红外光谱表现出其氨基酸组成特征,与猪血ＳＯＤ存在差异．该酶在不同的升温速率下及经不同浓度的Ｈ２Ｏ２处理后的稳定性与猪血ＳＯＤ不同．其氨基酸组成与不同来源的同类酶存在差异．     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Isolation and characterization of a new bacteriocin,termed enterocin M,produced by environmental isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> AL41

The new bacteriocin, termed enterocin M, produced by Enterococcus faecium AL 41 showed a wide spectrum of inhibitory activity against the indicator organisms from different sources. It was purified by (NH₄)₂SO₄ precipitation, cation-exchange chromatography and reverse phase chromatography (FPLC). The purified peptide was sequenced by N-terminal amino acid Edman degradation and a mass spectrometry analysis was performed. By combining the data obtained from amino acid sequence (39 N-terminal amino acid residues was determined) and the molecular weight (determined to be 4 628 Da) it was concluded that the purified enterocin M is a new bacteriocin, which is very similar to enterocin P. However, its molecular weight is different from enterocin P (4 701.25). Of the first 39 N-terminal residues of enterocin M, valine was found in position 20 and a lysine in position 35, while enterocin P has tryptophane residues in these positions.     

Purification and characterization of multiple glutathione S-transferase isozymes from Chironomidae larvae.

Glutathione S-transferase (GST) has been implicated in the process of biotransformation of polycyclic aromatic hydrocarbons and of other organic pollutants by Chironomidae larvae. We have purified and characterized GST from cytosolic fractions of Chironomidae larvae. GST with an M(r) of 23 kDa has been purified to homogeneity from larvae by centrifugation, size exclusion chromatography on Sephadex G25, and glutathione affinity and anion exchange chromatography. The purified enzyme exhibited moderate activity towards 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, 4-nitropyridine-N-oxide, p-nitrobenzyl chloride, ethacrynic acid, and cumene hydroperoxide. The enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 5.5. The enzyme had a maximum activity at approximately pH 8 and showed activity between 30 and 40 degrees C. It became inactive at higher temperature (>50 degrees C) for 5 min. The N-terminal sequence analysis of the amino acids shows a high % of conserved regions in the enzyme. The enzyme activity was comparable to levels of metabolism observed by animal GST involved in the detoxification of xenobiotics.     

A new type of mitogenic factor produced by Streptococcus pyogenes.

A new type of mitogenic factor (protein) was purified from the culture supernatant of a strain of Streptococcus pyogenes by SP-Sephadex C-25 column chromatography, preparative isoelectric focusing and reversed-phase high-performance liquid chromatography. The purified factor, showing marked mitogenic activity in rabbit peripheral blood lymphocytes, gave a single-band staining for protein on SDS-PAGE. The molecular weight of the purified mitogenic factor was determined to be 25,370, which was different from those calculated from reported amino acid sequences deduced from 4 different nucleotide sequences of 3 kinds of streptococcal pyrogenic exotoxins (two SPEAs, SPEB and SPEC). The amino acid sequence of the N-terminal region of the purified mitogenic factor was determined to be Gln-Thr-Gln-Val-Ser-Asn-Asp-Val-Val-Leu-Asn-Asp-Gly-Ala-Ser-Lys-Tyr-Leu- Asn-Glu - Ala-, which was also different from the reported N-terminal sequences deduced from the 4 different nucleotide sequences. These data indicate that this mitogenic factor is distinct from the already described streptococcal pyrogenic exotoxins.     

Purification to apparent homogeneity of a thymocyte specific growth factor from calf thymus

A thymic peptide previously found to recruit thymocytes from G1 into S phase has been purified from a crude thymic extract by subsequent steps of gel exclusion chromatography and reverse phase high performance liquid chromatography (HPLC). The purified material, which appeared homogeneous on thin-layer chromatography and HPLC, stimulated the DNA synthesis of cultured guinea pig thymocytes in a nanomolar concentration range. The amino acid composition revealed a high content of acidic amino acids and no apparent homology to previously defined growth factors and thymus differentiation hormones.     

金属螯合亲和层析法纯化猪红细胞超氧化物歧化酶

本文报道以Sephadex G-200为基质的金属螯合亲和层析法纯化SOD、其比活为4508U/mg蛋白,收率为90.4％,经酸、碱、SDS-PAGE、考马斯亮兰染色均呈一条带,特异性的SOD活性染色呈阳性。分子量为34,000,氨基酸组成测定表明Tyr含量少,Gly含量高,紫外吸收光谱最大吸收在258nm处,园二色谱结果表明SOD空间结构为β—折迭及无规态,含极少量或几乎不含α—螺旋,等电聚焦电泳测定SOD有微不均一性,等电点分别为5.25,5.35和5.65。     

Investigation of the correlation between charge and glycosylation of IgG1 variants by liquid chromatography–mass spectrometry

A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.     

A comparison of the structure and properties of serum transferrin from 17 animal species

1. A comparison of the chemical and physical properties of the iron transport protein transferrin, purified from the following seventeen animal sera, is reported; human, rhesus monkey, dog, cat, rabbit, guinea pig, mouse, rat, cow, sheep, goat, horse, pig, turkey, duck, turtle and rattlesnake. 2. Similarities and differences in molecular weight, isoelectric point, antibody specificity, effect of pH on iron release, number of sialic acid residues, amino acid composition and the N-terminal amino acid residue, are discussed. 3. The results are compared with the commonly accepted evolutionary origins of the 17 species.     

Extraction and purification of tissue phosphopeptides

We describe a method of extraction and partial purification of phosphopeptides isolated from pig brain or from the electrical organ of Torpedo marmorata. The extraction of the phosphopeptides was achieved by alcoholic 0,04 N potassium hydroxyde solution or by 10(-1) M KCL containing 10(-3) M EDTA and 10(-4) M DTT. After having tried various fractionation methods like ion exchange chromatography or gel filtration we chose chromatography on DEAE Sephadex followed by purification of the isolated fractions by Sephadex G 25 filtration. The most important phosphate fractions (one was purified to about 90 per cent) were characterized by the determination of the N/P ratio which was different from one phosphopeptide to another. The amino acid composition showed a high glycin, serine and "acid" amino acid content.The presence of phosphoserine was shown by electrophoresis and chromatography of a partial hydrolysate of in vivo 32P labelled phosphopeptides isolated from rat liver. The polyanionic structure of these phosphopeptides allow them to act as real ion exchangers which might be involved during active transport mechanisms in cellular membranes.