蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

The abaxial epiderm of Vicia faba L. was chosen to prepare the crude extract of ABA binding protein (ABA-BP). The activity of ABA-BP was dependent on different extraction methods. The specific binding capacity of ABA-BP extracted with 0.5% Triton X-100 (binding activity (B) = 0. 487 nmoL/g protein) was higher than that with extracted cold acetone (0. 325 nmol/g protein) or (NH4)2SO4(0. 223 nmol/ g protein). The activity duration of ABA-BP extracted with Triton X-100 (60% Bmax after 40 h) was longer than that with cold acetone (30% Bmax after 10 h). The ABA-BP activity of binding ABA was sensitive to pH change with an optimum pH of 6.5. The ABA-BP specific binding activity decreased quickly under high concentration of NaC1 ( > 300 mmoL/L), but increased 12% with 5 mmol/L KC1. Some divalent cations like Ca2+ and Mg2 + were required for enhancing the ABA-BP activity. These optimum conditions are primordial for ABA-BP purification.     

Subcellular Localization and Kinetic Characterization of ABA Binding Proteins in Maize Root

By means of differential centrifugation, cytosol fraction and microsome were prepared from maize roots which have been grown in dark for 4 d. Highly purified plasma membranes were isolated from the microsome in two-phase aqueous system which is composed of 6.9 % (W/W) Dextran T500 and PEG 3350. The tonoplast was collected from the interface between 1% and 8% (W/W) Dextran T70 after gradient centrifugation. Electron microscopic observation and marker enzyme activities analysis proved that these fractions contained very few other membranes. Microvolume radioactivity ligand binding assay indicated that the specific binding sites of ABA in maize root microsome were mainly distributed on tonoplast and plasma membrane fractions. Their specific binding activity was 2485.4 and 1257.3 fmol/mg protein, respectively, the specific binding activity of cytosol fraction being the lowest (one order of magnitude lower). The dissociation constant (KD) of ABA-BP in plasma membrane was 1.57 nmol/L.     

蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

以蚕豆（ＶｉｃｉａｆａｂａＬ．）叶片下表皮为材料,比较ＴｒｉｔｏｎＸ１００、冷丙酮和（ＮＨ４）２ＳＯ４对ＡＢＡ结合蛋白（简称ＡＢＡＢＰ）的提取效果。结果表明：０．５％（Ｗ／Ｖ）ＴｒｉｔｏｎＸ１００去垢剂提取的ＡＢＡＢＰ与ＡＢＡ特异结合活性较高（０．４８７ｎｍｏｌ／ｇｐｒｏｔｅｉｎ）,维持结合活性的时间较长（４℃下反应４０ｈ保持最大结合的６０％）;而冷丙酮法提取的ＡＢＡＢＰ特异结合活性只有０．３２５ｎｍｏｌ／ｇｐｒｏｔｅｉｎ,且容易失活,１０ｈ仅保持最大结合的３０％左右。实验比较了各种盐离子对ＡＢＡＢＰ的影响,高盐（＞３００ｍｍｏｌ／ＬＮａＣｌ）不利于ＡＢＡＢＰ的结合反应,低浓度ＫＣｌ对ＡＢＡＢＰ活性略有促进。ＡＢＡＢＰ的结合活性需要介质中有一定量的Ｃａ２＋和Ｍｇ２＋,用ＥＤＴＡ螯合介质中Ｍｇ２＋、Ｃａ２＋后,ＡＢＡＢＰ活性大大降低,分别为最大结合的７５％和６０％。ＡＢＡＢＰ与ＡＢＡ反应的最适ｐＨ在６．５,这些条件为亲和层析纯化ＡＢＡＢＰ提供了依据。     

Purification of Maize Cytosolic 70 kD Stress Protein: a Calmodulin-binding Protein in Plants

The maize cytosolic 70 kD stress protein (HSC70) has been purified by a two-step procedure employing affinity chromatography on ATP-agarose followed by DEAE52 ion-exchange chromatography. Using a biotinylated cauliflower calmodulin (CAM) gel-overlay technique in the presence of 1 mmol/L Ca2+ , the HSCT0 could bind to CAM. No band was shown on sodium dodecyl sulfate-polyacrylamide gel overlayed with biotinylated cauliflower CaM when 1 mmoL/L Ca2+ was replaced by 5 mmol/L EGTA. It indicated that the binding of HSC70 to CaM was dependent on Ca2+. The purified HSC70 inhibited the activity of CaM-dependent NADK and the degree of inhibition increased with augmentation of the HSC70, which appeared to be typically characteristic to CaM- binding protein.     

人神经生长因子融合蛋白的纯化及其生物活性的研究

用一株基因工程菌Ｅ．ｃｏｌｉ２４２６／ｐＭＮ表达了麦芽糖结合蛋白－人神经生长因子融合蛋白．菌体超声破碎后,上清液经直链淀粉亲和柱一步即可获ＳＤＳ－ＰＡＧＥ纯融合蛋白（５５ｋＤ）,为麦芽糖结合蛋白（４２ｋＤ）与人神经生长因子（１３ｋＤ）的络合物,产率约１０％．产物用鸡胚背根神经节检测生物活性,１ＢＵ不大于１０ｎｇ,与小鼠颌下腺β神经生长因子具有类似生物活性     

苹果果实细胞质中依赖活体组织的ABA高亲和力特异结合蛋白

将苹果（Malus pumila L.cv.Starkrimon)果肉微粒体和细胞可溶组分在含有^3H-ABA的缓冲介质中分别温育,仅在细胞可溶组分中测到微弱的^3H-ABA结合活性。但是,如何将果肉组织圆片在^3H-ABA缓冲介质中直接温育,经制备亚细胞组分后直接测定,在细胞可溶组分中测到很高的^3H-ABA特异结合活性。果肉圆片用沸水预先热处理使细胞可溶组分中的^3H-ABA结合活性完全丧失,说明ABA结合依赖于组织的活体状态。药理实验证明了ABA结合位点的蛋白质性质,同时证明该蛋白的活性中心具有－SH和丝氨酸基因。ABA结合蛋白对ABA的结合具有可饱和性、可逆性和高亲和力。Scatchard作图证明存在2种ABA结合蛋白,一种具有较高的亲和力,其解离常数（Kd)为2.9mmol/L,另一种亲和力相对较低,其Kd值为71.4nmol/L。用ABA结构相似物进行的竞争实验证明了ABA结合蛋白对配体结合的立体特异性。分析了ABA结合蛋白与ABA结合的时间曲线、pH和温度依赖性。本研究检测到的依赖活体组织的ABA结合蛋白可能是果实发育过程中介导ABA信号的受体。     

In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits (in English)

The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd =2.9 nmol/L) and the other with lower affinity ( Kd =71.4 nmol/L). Phaseic acid, 2- trans -4- trans -ABA or cis-trans -(－)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.     

两步串联层析法纯化鼠抗人CD80单克隆抗体4E5

采用阴离子交换与凝胶过滤两步串联层析法,纯化了小鼠腹水来源的CD80阻断型单克隆抗体4E5。腹水样品经离心、过滤预处理后,在Tris-HCl缓冲溶液(pH8.0, 50mmol/L)条件下上阴离子交换柱对目的单抗进行捕集,采用0-0.5 mol/L NaCl浓度分步洗脱;含目的单抗的洗脱馏分再上凝胶过滤柱纯化,用PB缓冲溶液（pH7.2, 20mmol/L）洗脱,获得目的单抗4E5,其生物学活性高、纯度大于95%,抗体总回收率达61%。     

玉米根ABA结合蛋白的亚细胞定位及动力学性质

以玉米（Ｚｅａ ｍａｙｓＬ．）根或胚芽鞘为材料,经匀浆、分级离心得到胞质部分和膜部分（微粒体）,进一步用６．２％ （Ｗ／Ｗ ） Ｄｅｘｔｒａｎ Ｔ５００ 和ＰＥＧ ３３５０ 两相系统制备质膜,用１％ 和８％ （Ｗ ／Ｗ） Ｄｅｘｔｒａｎ Ｔ７０ 梯度离心制备液泡膜． 电镜鉴定及多种标志酶检测表明,制备获得了高纯度正向型质膜和富含液泡膜的组分,其它内膜的污染很少． 用微量放射配体结合（ＭＲＬＢ）实验证明,玉米根微粒体的ＡＢＡ专一性结合位点主要分布在液泡膜和质膜上,这两种膜组分与ＡＢＡ 的特异结合活性分别为２４８５．４ ｆｍ ｏｌ／ｍ ｇ ｐｒｏｔｅｉｎ 和１２５７．３ ｆｍ ｏｌ／ｍ ｇ ｐｒｏ－ｔｅｉｎ,玉米根段胞质部分结合活性最低（差一个数量级）．质膜上ＡＢＡ－ＢＰ与ＡＢＡ 的结合平衡解离常数（ＫＤ）为１．５７ ｎｍ ｏｌ／Ｌ．     

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化

对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）高效表达克隆ｐＺＷ．ＧＭ的表达产物进行了纯化,并对纯化的ＧＭ－ＣＳＦ进行了Ｎ端氨基酸序列分析。人ＧＭ－ＣＳＦ基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过超声破菌、包涵体抽提、凝胶过滤层析、复性、离子交换一系列化步骤,终产物纯度达９９％,按蛋白总量计算回收率达１０％,比活性达１×１０＾７ｕ／ｍｇ蛋白质。通过测定纯化人ＧＭ－ＣＳＦ的Ｎ端１     

Purification and characterization of the human platelet alpha 2-adrenergic receptor

Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.     

Purification and partial characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase produced by<Emphasis Type="Italic">Thermomonospora fusca</Emphasis>

Thermomonospora fusca produced a relatively high level of alpha-L-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparent Km value for the crude alpha-L-arabinofuranosidase preparation was 180 mumol/L 4-nitrophenyl alpha-L-arabinofuranoside; the upsilon lim value was the release of 40 mumol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of approximately 92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular alpha-L-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified alpha-L-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.     

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。     

牛脑中神经特异性S-100蛋白的纯化及鉴定

牛脑充分匀浆后经三次硫酸铵分级沉淀,再通过一次DEAE-Sepharose CL-6B层析柱,线性梯度洗脱后共收集4个峰洗脱液。PAGE分析(7.5％凝胶)显示第3峰为单一区带;免疫双扩散证实该洗脱液中蛋白为S-100蛋白。SDS-PAG E分析显示S-100蛋白分子量约为10kD;非还原条件下,凝胶过滤(Sephadex G-75)显示S-100蛋白位于MW为20kD区域。认为该纯化方法简便、快速,可获得较高纯度的S-100蛋白,活性高达1∶128以上,完全能满足进一步研究之用。     

Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.     

Purification and characterization of fatty acid-binding protein from rat kidney

We detected the presence of a fatty acid-binding protein (FABP) in rat kidney cytosols. This protein was eluted and purified 9.3-fold by sequential gel filtration and anion-exchange chromatography. Homogeneity was shown by a single band on polyacrylamide gel with a molecular weight of about 15,500. It had an optimum binding pH of 7.4. The binding of palmitate to the protein was saturable. Examination of fatty acid binding revealed the presence of a single class of fatty acid-binding sites. The apparent dissociation constant was 1.0 microM and the maximal binding capacity was 48 nmol/mg of protein. This protein showed similar binding characteristics for palmitate, oleate, and arachidonate. Rabbit antibody to this cytosolic FABP gave a single precipitin line with the antigen and selectively inhibited [14C]palmitate binding to the protein.     

小麦面粉Puroindoline蛋白的提取与纯化

Puroindoline蛋白是小麦面粉中一种非常重要的蛋白质,不仅影响和决定了籽粒的硬度,而且有抗G^＋、G^-菌以及抗真菌的作用。用含4％TritonX-114、100mmol／L pH7．8Tris-HCl缓冲液处理小麦面粉来分离Puroindoline蛋白。经处理后得到的蛋白质混合溶液首先用分子筛葡聚糖G-75纯化,每个收集管内的组分经SDS-PAGE分析,分子量小于31kD的蛋白质组分被回收和集中,回收的蛋白质组分经PEG20000浓缩后,再用离子交换柱羧甲基纤维素（CM-23）进行纯化。其洗脱液分别是双蒸水和NaCl,梯度为0．05～0．7mol／L、8mmol／L pH5．5的MES缓冲液,回收只含15kD的蛋白质的组分,接着用PEG20000浓缩。最后冷冻干燥得到Puroindoline蛋白。     

重组人胰岛新生相关蛋白的表达、纯化及免疫活性研究

摘要 目的：对胰岛新生相关蛋白（Islet neogenesis associated protein ,INGAP）进行表达、纯化,并检测其免疫活性。方法： INGAP基因片段插入表达载体pET22b(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,Heparin Agrose亲合柱层析为第一步纯化,Superdex75凝胶过滤层析作为第二步精细纯化,HPLC测定INGAP蛋白的浓度,将纯化的INGAP蛋白经注射途径免疫家兔,制备兔抗INGAP血清,采用免疫双扩、ELISA及Western Blot分析INGAP的免疫活性。结果INGAP以包涵体形式表达,表达产量高达总菌体蛋白的40%左右,经Heparin Agrose亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.81%,表达及纯化的INGAP具有良好的免疫活性。