重组人胰激肽原酶复性工艺的研究

目的:研究重组人胰激肽原酶包涵体变性及复性的工艺。方法:对本实验室构建的重组人胰激肽原酶大肠杆菌进行IPTG诱导表达表达成功后,菌体经超声破碎释放包涵体,包涵体经洗涤、变性、稀释和尿素梯度凝胶过滤色谱这两种方法复性后(Sephadex-G75),通过测定酶活检验复性效果。结果:①重组人胰激肽原酶工程菌经过IPTG诱导后能够表达目的蛋白,目的蛋白以包涵体形式存在,将细胞破碎后,包涵体经过3次洗涤,纯度达到71.93%;②变性包涵体经24小时稀释复性后,蛋白浓度达到72.61μg/m L,酶的比活达到13.84 U/mg;③变性包涵体经过2个小时的尿素梯度凝胶过滤复性后,蛋白浓度可达到830.07μg/mL,酶的比活达到48.61 U/mg。结论:两种复性方法均可以使包涵体达到一定的浓度和比活,比较发现尿素梯度凝胶过滤色谱具有复性时间短和比活力高等优点,可作为重组人胰激肽原酶复性的一种有效的手段。     

重组N-乙酰鸟氨酸脱乙酰基酶包涵体的稀释复性

N-乙酰鸟氨酸脱乙酰基酶（N-Acetylornithine deacetylase,NAO）是一种重要的用于手性拆分的酶,具有广泛的底物选择性,常用于多种活性氨基酸的酶法拆分。采用稀释复性法研究了重组NAO包涵体的复性条件,如蛋白浓度、复性液中尿素浓度、pH、GSH浓度及c（GSH）/c（GSSG）比例,同时对稀释操作方式进行了考察,得到了较为适宜的复性条件。结果表明,尿素能有效抑制复性过程中蛋白质的聚集,随着蛋白质浓度的增加,复性效果变差。当复性缓冲液中尿素浓度为2 mol/L,GSH浓度为5 mmol/L,c（GSH）/c（GSSG）为2.5,pH为8.5,在4℃下进行分批稀释复性操作,复性后重组NAO的活性为1.077 U/mL,比酶活达到14.943 U/mg,与可溶性表达的NAO比较,复性率达到21.48%。     

甲胎蛋白的原核表达及复性优化

构建甲胎蛋白的原核表达载体p ET32a-AFP,对包涵体形式表达的甲胎蛋白进行复性优化。将构建的重组质粒p ET32a-AFP转化入E.coli,IPTG诱导表达后,经亲和层析纯化获得AFP包涵体,通过对复性过程、p H、添加剂等的研究摸索,获得最佳复性条件。当采用添加0.5 mol/L L-精氨酸的一步法透析复性方法,且透析液p H值为8.5,重组人AFP包涵体蛋白起始浓度为1.0 mg/m L时,复性效率最高。该复性方法获得蛋白质具有较高的回收率且操作简便。     

融合牛肠激酶轻链EKL包涵体蛋白的复性纯化

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。     

融合牛肠激酶轻链EKL包涵体蛋白的复性纯

大肠杆菌高密度发酵表达肠激酶轻链融合蛋白DsbA-rEK_L,主要以包涵体形式存在。包涵体经4mol/L尿素和 0.5% Triton X100洗涤,以6mol/L盐酸胍、100mmol/L DTT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/L CaCL2后快速自切。经IDASepharose及Qsepharose 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rEK_L,每升发酵液经复性及纯化后,可得rEK_L 60mg/L以上,使以融合蛋白表达rPA等药用蛋白成为现实。     

从重组融合蛋白包涵体中释放大环肽过程优化

[目的]探索不同条件对包涵体复性的影响,优化重组融合蛋白包涵体中释放大环肽的工艺条件。[方法]对纯化后的包涵体进行不同变性液、透析方式及是否超声破碎处理的比较;并在此基础上探讨不同透析温度、谷胱甘肽浓度与L-Arg浓度对大环肽产量的影响。采用Tanon凝胶分析系统与紫外吸收法对大环肽的产量进行分析。[结果]使用8 mol/L的尿素溶解包涵体后,获得的上清液在25℃条件下,在含有1 mmol/L GSH、0.1 mmol/L GSSG、0.8mmol/L L-Arg的透析液中进行梯度透析,其得率为每克包涵体获得约1.4 mg大环肽。[结论]成功从包涵体中获得大环肽并优化工艺条件。     

大肠杆菌表达的人IL-6复性条件研究

采用稀释法复性,筛选重组人白细胞介素-6(rhIL-6)包涵体蛋白的复性条件。结果显示,复性液的浓度、pH值、复性时间和复性蛋白浓度,对重组人白细胞介素-6复性效果有很大影响。在复性液为2mol/L尿素、pH8.5、复性时间为24h和复性蛋白浓度50μg/ml条件下,重组人白细胞介素-6包涵体蛋白的复性效果最佳。     

重组人脱氧核糖核酸酶Ⅰ的复性及活性鉴定

建立尿素梯度凝胶过滤复性重组人脱氧核糖核酸酶Ⅰ的方法。将诱导表达的重组人脱氧核糖核酸酶Ⅰ包涵体通过初步纯化后变性,然后在尿素梯度凝胶过滤色谱柱Sephadex G-75中复性,洗脱流速0.4 mL/min,复性完毕后透析除去小分子复性剂,使用琼脂糖电泳法检验其有活性后,再用单向酶扩散法测定其酶活力为655.8 U/mg,复性得率为83.7%。最后通过LC-ESI-MS/MS从氨基酸序列组成上证明复性产物是重组人脱氧核糖核酸酶Ⅰ。结果表明,建立的方法能成功用于复性变性的重组人脱氧核糖核酸酶Ⅰ包涵体蛋白,获得了可用于结构和功能研究的具有生物学活性的重组人脱氧核糖核酸酶Ⅰ。     

从包涵体中制备重组人血管抑素

目的:研究从包涵体中制备重组人血管抑素的可行工艺。方法:采用发酵法制备表达重组 人血管抑素的大肠杆菌,经离心、破碎、洗涤后得到包涵体,以盐酸胍作为变性液溶解包涵体,经 稀释透析法复性重组人血管抑素,以SDS-PAGE及Wetern-blot鉴定纯度,T法鉴定其生物活性 MT。结果从6．88g包涵体中得到了322．5mg的重组人血管抑素,得率4．7％。电泳鉴定为单一条 带,在浓度0．1mg/ml时,对内皮细胞(HEMC-1)最高抑制率可达33％。结论:得到了一条可行的 血管抑素的制备工艺。     

重组血管内皮细胞生长因子包涵体复性条件研究

利用大肠杆菌ＢＬ２１（ＤＥ３） 表达血管内皮细胞生长因子１６５（ ＶＥＧＦ１６５） ,表达蛋白以包涵体形式存在。为了获得大量有生物活性的蛋白质,我们对影响复性的参数：氧化型、还原型谷胱甘肽比例,复性液的ｐＨ 值,复性时间,精氨酸浓度以及血管内皮细胞生长因子（ ＶＥＧＦ１６５） 包涵体的起始浓度进行了较为系统的研究,初步获得了具有一定生物活性的ＶＥＧＦ１６５ 二聚体蛋白。     

On-column refolding of an insoluble His6-tagged recombinant EC-SOD overexpressed in Escherichia coli

The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a( ) and transformed into E. coli BL21 (DE3). The corresponding protein that was overexpressed as a recombinant His6-tagged EC-SOD was present in the form of inactive inclusion bodies. This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4:1. The purified enzyme was active,showing that at least part of the protein was properly refolded. The protein was made concentrated by ultrafiltration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A280 profile.Based on the results of electrophoresis, we concluded that the two fractions were formed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of the dimer was much higher than that of the single monomer. The special activities of the two fractions were found to be 3475 U/mg protein and 510 U/mg protein, respectively.     

In vitro activation, purification, and characterization of Escherichia coli expressed aryl-alcohol oxidase, a unique H2O2-producing enzyme

Aryl-alcohol oxidase (AAO), a flavoenzyme with unique spectral and catalytic properties that provides H2O2 for fungal degradation of lignin, has been successfully activated in vitro after Escherichia coli expression. The recombinant AAO (AAO*) protein was recovered from inclusion bodies of E. coli W3110 transformed with pFLAG1 containing the aao cDNA from Pleurotus eryngii. Optimization of in vitro refolding yielded 75% active enzyme after incubation of AAO* protein (10 microg/ml) for 80 h (at 16 degrees C and pH 9) in the presence of glycerol (35%), urea (0.6 M), glutathione (GSSG/GSH molar ratio of 2), and FAD (0.08 mM). For large-scale production, the refolding volume was 15-fold reduced and over 45 mg of pure active AAO* was obtained per liter of E. coli culture after a single anion-exchange chromatographic step. Correct FAD binding and enzyme conformation were verified by UV-visible spectroscopy and circular dichroism. Although the three enzymes oxidized the same aromatic and aliphatic polyunsaturated primary alcohols, some differences in physicochemical properties, including lower pH and thermal stability, were observed when the activated enzyme was compared with fungal AAO from P. eryngii (wild enzyme) and Emericella nidulans (recombinant enzyme), which are probably related to the absence of glycosylation in the E. coli expressed AAO.     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.     

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

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     

血管内皮细胞生长因子复性条件研究

利用大肠杆菌 BL2 1 (DE3)表达血管内皮细胞生长因子 (VEGF16 5)表达蛋白以包含体形式存在。为了获得有生物活性的蛋白质 ,我们对影响复性的参数 :氧化型、还原型谷胱甘肽比例 ,复性液的 p H值 ,复性时间 ,精氨酸浓度 ,血管内皮细胞生长因子起始浓度进行了较为系统的研究 ,初步获得了具有一定生物活性的 VEGF16 5二聚体蛋白。     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.