共查询到20条相似文献,搜索用时 24 毫秒
1.
2.
3.
Recombinant Human Ribosomal Protein S16: Expression,Purification, Refolding,and Structural Stability
The cDNA of human ribosomal protein S16 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. It was found that 21 +/- 4% of the amino acid sequence of the protein forms alpha-helices and 24 +/- 3% is in beta-strands. The protein structure stability was studied at various pH values and urea concentrations. The protein is quickly denatured at pH above 8.0, whereas increasing of urea concentration causes slow unfolding of the protein. 相似文献
4.
重组人尿激酶原的分离纯化及性质研究 总被引:1,自引:0,他引:1
本文报道从人尿激酶原(pro-urokinase,pro-UK)基因重组工程菌E.coliJA221表达产物的复性液中纯化尿激酶原的方法。复性产物经Zn^2+选择性沉淀,尿激酶抗体亲和柱层析,benzamidine-Sepharose CL 4B亲和吸附,即得比活达110000IU/mg的纯化产品。样品经SDS-PAGE鉴定,在还原及非还原条件下,均表现为分子量为43kd的单一条带。动力学研究测得 相似文献
5.
大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEKL,主要以包涵体形式存在。包涵体经4mol/L尿素和0.5%TritonX-100洗涤,以6mol/L盐酸胍、100mmol/LDTT溶解,在胱氨酸存在下,以脉冲加样方式复性。融合蛋白复性在6mmol/L胱氨酸存在下、脉冲加量0.03mg/mL和复性终蛋白浓度0.3mg/mL为最佳复性方案。复性的融合蛋白加2mmol/LCaCL2后快速自切。经IDA-Sepharose及Q-Sepharose纯化,rEKL纯度可达95%以上,可高效酶切重组瑞特普酶融合蛋白Trx-rPA。实现了大规模生产rEKL,每升发酵液经复性及纯化后,可得rEKL60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。 相似文献
6.
融合基因EGF-IL-18与表达载体pET32a( )连接构建融合型原核表达质粒pET32a( )-EGF-IL-18。该基因在E.coliRosetta(DE3)中获得高效表达,SDS-PAGE分析表明表达产物大部分以包涵体形式存在。以2mol/L尿素和1%TritonX-100对包涵体进行反复洗涤后,利用离子交换柱层析对包涵体进行柱上复性,结果表明离子交换层析柱上复性不仅能够获得可溶性的EGF-IL-18融合蛋白,而且产物同时得到纯化,纯度大于90%。复性的EGF-IL-18经分子筛进一步纯化后,体外实验证明具有促进人外周血单个核细胞(PBMC)IFN-g基因表达的能力。该方法简单、高效,为进一步开展EGF-IL-18的动物实验及其大量纯化制备打下基础。 相似文献
7.
8.
重组尿激酶原的纯化和性质研究 总被引:3,自引:0,他引:3
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. 相似文献
9.
10.
The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in the inclusion body in E. coli. The recombinant bacteria were fermented to high density, with high expression of the fusion protein. After being washed with 0.5 % Triton X-100 and 4 mol/L urea, the inclusion body was dissolved in 6 mol/L guanidine and 100 mmol/L DTT, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03 mg/mL of fusion protein until its final concentration reached 0.3 mg/mL. The refolded protein was autocleaved, and the active EKL molecule was released after the addition of 2 mmol/L of CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95 %, with a high activity to cleave the recombinant reteplase fusion protein, Trx-rPA. The yield of purified rEKL was more than 60 mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large scale in a way such as expressed in the form of fusion proteins. 相似文献
11.
重组人白细胞介素2(rhIL2)基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过菌体发酵和收集、超声破菌、包涵体抽提、稀释透析初步复性、反相高压液相色谱纯化,终产物纯度大于99%。反相高压液相色谱不仅可以纯化rhIL2,且使产物的总活性提高79~9倍,比活性提高14~18倍,达到15×107u/mg蛋白质,蛋白得率为50%~56%。结果表明,反相高压液相色谱具有纯化和显著提高rhIL2折叠效率的双重功效,为非SDS变复性法生产rhIL2提供了简便有效的方法 相似文献
12.
重组包涵体蛋白质的折叠复性 总被引:48,自引:1,他引:48
冯小黎 《生物化学与生物物理进展》2001,28(4):482-485
综述了减少包涵体形成、包涵体分离和溶解以及包涵体折叠复性的策略及其最新进展 .详细讨论了包涵体蛋白质折叠复性的基本原则、包涵体折叠复性促进剂和包涵体折叠复性方法 相似文献
13.
Human granulocyte macrophage colony-stimulating factor (hGM-CSF) is a haematopoietic growth factor and proinflammatory cytokine. Recombinant hGM-CSF is important not only as a research tool but also as a biotherapeutic. However, rhGM-CSF expressed in E. coli is known to form inclusion bodies of misfolded, aggregated protein. Refolding and subsequent purification of rhGM-CSF from inclusion bodies is difficult with low yields of bioactive protein being produced. Here we describe a method for the isolation, refolding and purification of bioactive rhGM-CSF from inclusion bodies. The method is straightforward, not requiring extensive experience in protein refolding nor purification and using standard laboratory equipment. 相似文献
14.
自牛肝中纯化了蛋白二硫键异构酶(PDI),并对重组蛋白的酶促折叠过程进行了探讨.结果表明。在等摩尔PDl的催化作用下,可使1mg/ml的IIJ-2的正确折叠率提高到58%以上,比活性由4×106u/mg增加到8.2×106u/mg,PDl还能部分纠正二硫键错配的IL-2异构体成为正确折叠的lL-2和防止IL-2通过cys的链问交联形成聚合体。GM-CSF在PDl催化下也有类似的结果。PDl作用的关键是它所催化的琉基一二硫键的交换反应。 相似文献
15.
收集rhbFGF工程菌菌体,包涵体纯化后经凝胶过滤柱复性,所得复性后bFGF通过肝素亲和柱纯化并测活。各种复性方案中复性后bFGF比活最高为2.5×105U/mg,得率最高为74%。复性液中的变性剂浓度及是否含有还原剂,柱流速及柱体积都会影响复性后bFGF的得率和活性。 相似文献
16.
棉铃虫单核衣壳核型多角体病毒(HaSNPV)是我国第一个商品病毒杀虫剂,具有使用安全、害虫不产生抗药性等优点,是一种很有发展潜力的生物农药。幼虫虫体受病毒感染后,HaSNPV几丁质酶在其液化过程中起了很大的作用,因此可以作为增效剂以显著提高细菌、病毒、真菌等微生物杀虫剂的毒力,并具有更高的安全性。将HaSNPV几丁质酶基因构建到原核表达载体pET28a中,经测序检验后转化至大肠杆菌Rosetta,然后以IPTG作为诱导剂,目标蛋白以包涵体的形式得以成功表达。在变性条件下,包涵体经镍 次氮基三乙酸(Ni-NTA)柱层析纯化,并以两种不同的方法进行复性,均可获得具有活性的HaSNPV几丁质酶。 相似文献
17.
重组类胰岛素样生长因子-Ⅰ的纯化与复性 总被引:3,自引:0,他引:3
目的 获得高纯度和高活性的胰岛素样生长因子(Insulin-like growth factor, IGF-1);方法 构建好的BL21大肠杆菌工程菌经IPTG诱导,以融合一段截短型半乳糖苷酶及His-tag形式表达IGF-1融合蛋白(约15,000Da),超声破碎,提取包涵体经镍柱亲和层析后, 用羟氨切割纯化的融合蛋白,纯化后的蛋白质在小分子保护剂及GSH/GSSG的存在下复性。结果 经Ni2+柱亲和层析, IGF-1纯度达90%以上,复性后得到有较高生物活性的IGF-1。结论 IGF-1发酵及纯化和复性方法的建立为大量生产IGF-1打下了基础。 相似文献
18.
Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different
refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column
refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl
S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA
inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25
column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing
urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was
increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
Revisions requested 31 October 2005; Revisions received 20 December 2005 相似文献
19.
20.
One major bottleneck in protein production in Escherichia coli for structural genomics projects is the formation of insoluble protein aggregates (inclusion bodies). The efficient refolding
of proteins from inclusion bodies is becoming an important tool that can provide soluble native proteins for structural and
functional studies. Here we report an on-column refolding method established at the Berkeley Structural Genomics Center (BSGC).
Our method is a combination of an ‘artificial chaperone-assisted refolding’ method previously proposed and affinity chromatography
to take advantage of a chromatographic step: less time-consuming, no filtration or concentration, with the additional benefit
of protein purification. It can be easily automated and formatted for high-throughput process. 相似文献