多通道流动电泳技术及其在蛋白质、酶和抗体纯化中的应用研究

提出一种新型制备电泳方法即多通道流动电动电泳技术,建立了微机控制的电泳设备,考查了操作条件对设备分离效率的影响。应用该技术进行牛血清清蛋白(BSA)-牛血红蛋白(HBB)混合物的连续分离,每小时从含BSA、HBB备37．Omg的蛋白质混合物中分离出13．6mg的BSA和20．Omg的HBB。应用该技术进行尿激酶粗品的纯化,可将其比活力由4 000IU／mg提高到4×10⁴IU／mg以上．产量在10×10⁴IU／h以上。将该技术与(NH₄)SO- 沉淀方法结合,从15ml免疫鼠血清中分离出12．5mg高纯度尿激酶抗体IgG,产量为2．1mg／h。上述结果证明多通道流动电泳技术其有分离速度快、精度高和可操作性好等优点,在生物产品分离领域中具有广阔的应用前景。     

电泳亲和色谱技术分离蛋白质*

亲和色谱利用亲和配体与目标组分间的特异性结合作用实现对目标组分的纯化,该分离方法分辨率高．在生物物质的分析和分离领域得到日益广泛的应用[1]。亲和色谱在分离过程每一步操作中,液相主体中的溶质分子必须经过一系列扩散过程才能进入到固定相颗粒孔内完成吸附或解吸等质量置换反应,被置换出的物质再由颗粒内扩散出固定相颗粒进入流动相。与质量置换反应过程相比,扩散过程由于速度较慢而常成为亲和色谱分离过程的速度控制步[2]。开发新型介质以强化扩散过程成为近年来色谱分离技术研究的热点,典型成果有灌注色谱介质(Perfllsion clnromatogr;tph>r)13,引。基于制备电泳技术方面的研究成果[5.6],我们提出将多通道流动电泳与亲和色谱相结合形成一种新型制备规模的生物分离技术即电泳亲和色谱技术,其基本思想是利用电场强化扩散过程以加速分离过程的进行。我们的前期工作已证明该方法的可行性[7]。本文以人血清清蛋白(Human seguigt albllmm．以下简称has)和Blue Sepharose 6 Fkt F10wⅢ介质(以下简称Blue介质)为例,通过实验系统地考察电流强度对电泳亲和吸附和电泳洗脱过程的影响,并用电泳亲和色谱的方法纯化has。     

高分子量和低分子量尿激酶的分离纯化及动力学性质研究

人尿激酶粗品经苯甲脒亲和柱纯化和Protein-PahSP柱分离后,得到两种分子量的尿激酶（UK）,即高分子量尿激酶（HUK）和低分子量尿激酶（LUK）．采用考马斯亮蓝法测定蛋白质浓度,纤维蛋白平板法测定活力,测得HUK比活为2.9×105IU／mg蛋白,LUK为3.5×105IU／mg蛋白,活力回收为70％以上．经SDS-PAGE鉴定,HUK和LUK均是单一条带,分子量分别为54kD和33kD．HUK和LUK水解显色底物S2444的动力学常数,分别测得HUK的Km为64μmol/L,Kcat为15s-1,LUK的Km为49μmol／L,kcat为13s-1,LUK的催化效率（Kcat／Km）稍高于HUK．     

脉冲场凝胶电泳技术及其在真菌学研究中的应用*

脉冲电泳是用于分离大分子量DNA的一种电泳技术,已广泛用于真菌的核型分析,种群特异性鉴定,基因定位及遗传分析的研究。介绍了脉冲电泳的原理,发展和基本操作程序,并阐述了脉冲电泳技术在真菌分子生物学研究领域的应用。     

基于智能控制的微管电泳检测技术

国内外毛细管电泳(CE)产品深受电泳焦耳热存在的影响,使得毛细管电泳试样的流通量非常少,严重影响到了CE发展与应用。本文对毛细管电泳技术存在的问题进行进一步的量化分析,利用智能控制技术来构造微管电泳检测系统,采用内制冷的方式,及时原地的带走电泳过程中产生的焦耳热,采用多通道高压电源作为整个电泳系统的驱动力,通过对运行缓冲液的合理调控,实现对待分离组分的高效分离。研究出高效微管电泳技术的全分析系统模型,以期能给生命科学、材料科学、环境科学提供又一强有力的研究手段。     

一步法电泳分析肌联蛋白亚型和肌球蛋白重链

目的为研究超大分子量肌小节蛋白肌联蛋白(titin)的生理病理功能,在一次电泳过程中同时分离titin各亚型和中分子量肌小节蛋白肌球蛋白重链(myosin heavy chain,MHC)。方法使用16cm×18cm垂直电泳系统,在电泳板下1/3灌注10g/L SDS-PAGE胶,上2/3灌注60g/L SDS-琼脂糖(SDS-VAGE)胶。低温8℃下持续电泳5h,在电泳板上层以SDS-VAGE胶电泳分离titin亚型,下层以SDS-PAGE胶电泳分离MHC。电泳后VAGE胶使用银染法标记titin各亚型,PAGE胶使用考马斯亮蓝染色法标记MHC。结果 titin各亚型得到有效的分离,目标蛋白条带显示清晰,与其分子量大小一一对应,分离效果明确。结论一步法垂直电泳系统可应用于超大分子量蛋白的电泳,同时可分离多个分子量差距大的蛋白,提高蛋白电泳实验效率。     

表面改性的纳米Fe3O4颗粒用于抗血清快速分离纯化

采用硅烷化试剂Si(OC2H5)3C3H6NH2(APTES)对纳米Fe3O4颗粒表面进行氨基化改性后, 考察了不同浓度偶联剂戊二醛对于颗粒表面固定牛血清白蛋白(BSA)量的影响。此超顺磁性免疫铁颗粒(SPIO)加入兔抗BSA血清中特异性结合BSA抗体后, 用Gly-HCl缓冲液洗脱得到IgG。结果表明当戊二醛浓度大于10%时, 单位颗粒固定蛋白的量达到最大值约140 mg/mg, 10 min, 15 mg的SPIO即可将1 mL抗血清完全分离, 经过两次快速洗脱, 颗粒表面吸附的抗体即可得到纯化; 琼脂扩散实验表明分离后的抗体仍保持较高活性, SDS-PAGE电泳结果表明用此方法纯化后的兔抗BSA IgG纯度大于99%, 比传统的(NH4)2SO4法有了较大提高, 但纯化量并没有减少; SPIO在经过五次重复利用后仍能保持78%以上的分离效果。     

毛细管电泳在细菌分离分析中的应用

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

毛细管电泳在细菌分离分析中的应用*

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

尿激酶前体的纯化及其性质的研究

尿激酶前体是与尿激酶具有共同抗原性的一种新的纤溶酶原激活物。我们从人胎肾细胞条件培养液中提纯该物质。首先用抗UKIgG-Sepharose亲和层析得到尿激酶抗原相关蛋白,然后利用苯甲脒-Sepharose柱去除尿激酶获纯化的尿激酶前体。纯化倍数达930倍,得率为18％,所得尿激酶前体为单肽链结构蛋白质,分子量55kD,比活(11389IU/mg)低于尿激酶,二异丙基氟磷酸酯(DFP)不能抑制其活性。体外¹²⁵I-血凝块溶解试验表明尿激酶前体可特异地诱导血凝块溶解,对血浆纤溶系统无明显激活作用,血凝块溶解的时间曲线呈特征性“S”型。所得尿激酶前体是一种新的有别于尿激酶的纤溶酶原激活物。     

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.     

Construction and expression of antibody targeted plasminogen activator*

It has been known that antibody-mediated plasminogen activator will be much more specific than its parent molecular. To get a cheaper and more effective medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoclonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator. PCR was used to amplify the region of VL and VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu-PA-32KD(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via NdeI site. The recombinant protein was expressed in BL21 (DE3) plyS, a kind of E. coli. It was shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase. After partial purification: dialysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate. The specific activity was about 47,000 IU/mg, corresponding to 80,000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody. Activation of plasminogen by the chimera followed Michaelis-Menten kinetics, and the K_m was 1.08 uM.     

人源化鼠抗人纤维蛋白单链抗体-尿激酶融合蛋白在大肠杆菌中的功能性表达

尿激酶是目前临床上广泛使用的一种有效的溶栓制剂 ,但由于尿激酶缺乏对血栓的特异亲和性 ,导致临床上尿激酶的使用剂量较大 ,容易出现全身性出血等副作用。因而开发对血栓特异的导向溶栓制剂是目前溶栓药研制的一个重要方向。本实验室在以前的工作中 ,利用噬菌体表面呈现技术 ,筛选到一种对人纤维蛋白特异的鼠单链抗体[1 ] ,在对该鼠抗体可变区进行结构模建的基础上[2 ] ,对其进行了人源化改造和体外亲和力成熟 ,得到亲和力和特异性较鼠抗体更好的人源化单链抗体[3 ] 。为研制导向溶栓制剂 ,本实验室构建了人源化单链抗体 低分子量尿激酶的…     

Application of multichannel flow electrophoresis to separation of biomolecules: a survey

Multichannel flow electrophoresis (MFE) is a newly developed method for continuous separation of biological products at a preparative scale, In this short survey, the application of MFE in the separation of proteins, enzymes and antibodies are overviewed.     

Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.     

Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli

Thrombolytic therapy has been a major advance in thtreatment of myocardial infarction over the last twdecades. Urokinase (UK), streptokinase (SK), and tissuplasminogen activator (t-PA) are the common availablthrombolytic agents. These agents, however, hav…     

Continuous separation of proteins by poly(vinyl alcohol) shielded Multichannel Flow Electrophoresis

Summary A hydrophilic polysulphone membrane spaced multicompartment electrolyzer for Multichannel Flow Electrophoresis (MFE) was developed. Application of poly(vinyl alcohol) (PVA) in MFE resulted in a 35% increase of protein transmembrane flux. Continuous separation of bovine serum albumin (BSA) and haemoglobin bovine blood (HBB) model mixture by PVA shielded MFE yielded 56mg BSA and 48mg HBB per hour. The average recovery was 65%.     

Recovery of urokinase from integrated mammalian cell culture cryogel bioreactor and purification of the enzyme using p-aminobenzamidine affinity chromatography

An integrated product recovery system was developed to separate urokinase from the cell culture broth of human kidney cells HT1080. Supermacroporous monolithic cryogels provided ideal matrices with respect to surface and flow properties for use as cell culture scaffold as well as for affinity chromatographic capture step of the enzyme in the integrated system. The urokinase was produced continuously in the reactor running for 4 weeks with continuous circulation of 500 ml of culture medium. The enzyme activity in the culture medium reached to 280 Plough units (PU)/mg protein. Cu(II)-iminodiacetic acid (IDA)-polyacrylamide (pAAm) cryogel column was used to capture urokinase by integrating with the gelatin-coupled pAAm-cryogel bioreactor for HT1080 cell culture. After removing the urokinase capture column from the integrated system the bound protein was eluted. The metal affinity capture step gave 4.5-fold purification of the enzyme thus achieving a specific activity of 1300 PU/mg protein. The enzyme eluate from Cu(II)-IDA-pAAm cryogel capture column was further purified on benzamidine-Sepharose affinity column. This step finally led to a homogeneous preparation of different forms of urokinase in two different elution peaks with a best urokinase activity of 13 550 PU/mg of protein. As compared to initial activity in the cell culture broth, about 26.2- and 48.4-fold increase in specific activity was achieved with enzyme yields corresponding to 32% and 35% in two different peak fractions, respectively. Native electrophoresis and SDS-PAGE showed multiple protein bands corresponding to different forms of the urokinase, which were confirmed by Western blotting and zymography.     

Application of continuous zone electrophoresis to preparative separation of proteins

A comprehensive study of the application of continuous zone electrophoresis to preparative separation of proteins in free solution is presented. First, the influence of electric field strength, buffer residence time in the chamber, sample flow rate, and sample concentration on separation resolution and throughput were studied. Using multiple injections of sample into the electrophoresis chamber, a throughput of 500 mg protein/h was achieved for partially purified model proteins. Experiments on Escherichia coli crude extracts yielded a fivefold purification of beta-galactosidase along with a simultaneous separation of proteins from cell debris in a single step. Experiments correlating the electrophoretic mobility in continuous electrophoresis with the elution behavior in ion-exchange chromatography were performed on more than a dozen proteins which conclusively showed that separation of proteins in continuous zone electrophoresis is governed by net surface charge. Based on these results, the fraction numbers in which the proteins eluted could be correctly predicted. Proteins and enzymes with differences >0.5 M elution molarities in ion-exchange chromatography were separated by continuous zone electrophoresis on a preparative scale (mg/h or g/h) with >90% recovery. This corresponds to a preparative scale separation of proteins and enzymes which differ in apparent electrophoretic mobility by only 0.70 x 10(-5) cm(2)/V . s. (c) 1993 John Wiley & Sons, Inc.