可溶性TRAIL蛋白的高密度培养及补料策略研究

采用分批补料的方法高密度培养重组大肠杆菌C600/PbvTRAIL制备人可溶性TRAIL蛋白,优化发酵工艺,探索简单高效的分离纯化方法并测定蛋白生物活性。通过比较几种不同的补料策略：间歇流加、Dostat、pHstat,摸索了一种流加策略,即DOstatpHstat组合流加,有效的避免了发酵过程中,尤其是诱导表达阶段乙酸积累的增加,使TRAIL蛋白在高密度培养条件下,得到高效表达。菌体密度最终达到300g/L（WCW）以上,可溶性TRAIL蛋白占菌体总蛋白的4.2%,含量为1.1g/L。在整个发酵过程中,乙酸浓度接近于0,且未使用任何特殊手段,如纯氧、加压等,简化了发酵工艺,降低了发酵成本,为TRAIL的工业化生产创造了条件。     

TRAIL包涵体在细胞内重折叠现象的初步研究

肿瘤坏死因子相关凋亡诱导配体（Ap02L／TRAIL）是一种新型的抗肿瘤蛋白。但在大肠杆菌表达系统中,表达的TRAIL蛋白往往容易聚集形成包涵体。本文对重组TRAIL包涵体在胞内重折叠转化成可溶性TRAIL蛋白作了初步探索,结果表明：当蛋白表达后,迅速降低温度至30℃并添加50μg／mL氯霉素继续培养4h,可溶性TRAIL产量可提高至16．7％。这一结果在3．7L罐上也得到了证实,可溶性TRAIL表达量达到14．5％。     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

TRAIL蛋白的体外活性稳定性初步研究

大肠杆菌发酵表达的TRAIL蛋白经过两步纯化可得到纯度95%以上的活性蛋白,得率为40%.天然TRAIL蛋白为同源三聚体形式,在三聚体中心隐蔽结合Zn<'2+>,Zn<'2+>可维持TRAIL蛋白天然结构的稳定性.研究发现在纯化后的重组可溶性TRAIL蛋白中添加Zn<'2+>,并且Zn<'2+>与TRAIL单体的摩尔比...     

米曲霉中蓝藻抗病毒蛋白-N基因的克隆与表达

[目的]提高蓝藻抗病毒蛋白-N(CVN)的异源表达量,获得大量高纯度可溶性蛋白。[方法]采用RT-PCR从酱油发酵酱醪宏基因组中克隆获得CVN基因cvn-SF,构建了重组表达载体p ET32a-cvn-SF,转化E.coli BL21菌株,获得工程菌株,优化表达条件,通过Ni-NTA凝胶亲和层析对CVN-SF蛋白进行纯化。[结果]工程菌株在温度30℃、添加1 mmol/L的IPTG诱导剂、培养时间12 h的条件下获得最高表达量的可溶性蛋白。Ni-NTA凝胶亲和层析纯化得到了230.8 mg/L的CVN蛋白,占提取总蛋白含量的33.37%。[结论]所得CVN蛋白高于目前报道的CVN在大肠杆菌的最高表达量140 mg/L~([11])。     

提高大肠杆菌可溶性重组蛋白表达产率的研究进展

大肠杆菌表达重组蛋白相比真核细胞具有成本低廉、大规模发酵容易、条件易于自动化控制等优点,通过大肠杆菌表达重组蛋白是一种高效、经济的途径,重组蛋白表达量可达到大肠杆菌总蛋白质量的50%。具有正常生化活性的重组蛋白通常为可溶性形式,因而对于以得到活性产物(如抗体、酶等)为目的的研究,通常采用可溶性表达途径。目前已有多种以可溶性重组蛋白为活性物质的治疗性药物经批准上市,但并非所有外源基因均能实现可溶性高表达,因此重组蛋白的可溶性高表达具有重要研究价值。在总结近年提高经大肠杆菌可溶性表达重组蛋白产率研究的基础上,从启动子的选择、SD序列的引入、信号肽的优化、宿主细胞的选择、共表达其他蛋白质,高密度发酵等方面阐释在大肠杆菌中提高可溶性重组蛋白表达产率的方法。     

GABAA受体蛋白片段在大肠杆菌细胞表达条件研究

为了提高GABAA受体α1蛋白片段在大肠杆菌中表达量,研究了重组菌的发酵条件,包括培养基,接种量,温度,摇床转速,pH,诱导培养时间和诱导剂IPTG使用浓度等对GABAA受体蛋白片段表达的影响。结果表明重组菌以LB培养基为发酵基质,按3%接种量,37℃培养细胞3.5h后IPTG32℃诱导5h,菌体生物量为3.25g/L,目标蛋白表达量达95mg/L。用16L发酵罐进行放大培养,菌体生物量达4.95g/L,发酵周期5.5h。最高目标蛋白表达量达到136mg/L。     

Bacillus clarkii 7364 γ-环糊精葡萄糖基转移酶的可溶性表达及其催化特性分析

按照大肠杆菌偏爱密码子对Bacillus clarkii 7364来源的γ-环糊精葡萄糖基转移酶(γ-CGTase)进行优化,构建γ-CGTase原核表达菌株,摸索γ-CGTase的可溶性表达条件及纯化条件,并对其催化特性进行研究。结果表明:在28℃条件下实现了γ-CGTase的高效可溶性表达,可溶性蛋白占总蛋白表达量的63%,酶活可达3 830 U/mL。经(NH4)2SO4沉淀和α-CD-Sepharose 6B亲和柱纯化后,酶蛋白纯化了12.97倍,酶收率20.31%。使用该酶对木薯淀粉进行转化,转化产物中γ-环糊精(γ-CD)的比例可达90.9%,几乎无α-环糊精(α-CD),与天然酶的79%相比提高了15%。将该基因工程菌在20 L发酵罐中发酵,10 h后酶活达到4 375 U/mL,证实了其工业化放大的可能。该酶具有非常高的转化专一性,有非常好的工业化前景。     

重组尿酸氧化酶发酵工艺优化

目的:优化并获得重组尿酸氧化酶(rUOX)基因工程大肠杆菌BL21(DE3)/pET-32a-uox高密度发酵的工艺参数。方法:在三角摇瓶中进行培养条件的优化实验,分别考察了pH值、接种量、无机盐、碳源、诱导强度等对工程菌生长和重组蛋白表达的影响,得到了优化的发酵条件;在此基础上放大至NBS BIOFLO 110 14 L发酵罐,通过对诱导时机的优化,利用分批补料发酵的方式,使rUOX在高密度培养的条件下得到高表达。结果:在优化的发酵条件下,菌体密度(D600nm)最终达到50以上,相当于20 g/L干重;可溶性rUOX占菌体总蛋白量的45%,其含量达到3.45 g/L。结论:为规模化制备重组黄曲霉尿酸氧化酶奠定了基础。     

重组人白细胞介素-11工程菌的发酵条件研究

为了探讨发酵条件对大肠杆菌表达人白细胞介素-11融合蛋白的影响,利用正交实验设计,对工程菌的生长条件和人白细胞介素-11融合蛋白表达进行优化。在摇瓶中研究了培养基中的葡萄糖、蛋白胨、酵母抽提物的浓度、pH及摇床转速、装液量、接种量等。确定了工程菌生长及表达的培养基和培养条件:葡萄糖10g/L,蛋白胨20g/L,酵母抽提物10g/L,pH7.5,接种量10%,装液量10%,摇床转速220r/min及诱导时间为4~5h。然后在BiofloⅢ-5L发酵罐中以优化的发酵条件进行了3批实验,结果表明:工程菌量达到55g/L(DCW),重组人白细胞介素-11融合蛋白表达量为33%左右,为进行中试研究奠定了理论基础。     

Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying alpha-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

sTRAIL基因的表达、纯化及其生物学活性的初步研究

在原核表达系统中表达重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(sTRAIL)分子,纯化表达产物,并进行初步的生物学活性的研究中,通过提取外周血淋巴细胞总RNA进行RT—PCR克隆sTRAIL cDNA,构建sTRAIL的原核表达载体,用限制性酶切和DNA测序进行鉴定,IPTG诱导表达。以发酵罐放大培养,SDS-PAGE和Western-blot确认表达,用亲和层析和阳离子层析纯化表达产物,使用L929、Qay肝癌、K562人白血病等肿瘤细胞鉴定活性。结果发现重组表达的sTRAIL融合蛋白占总蛋白的39％,单纯蛋白纯化后纯度达95％,用抗人TRAIL多抗可以确认表达了TRAIL分子,能诱导几株肿瘤细胞凋亡。原核表达系统正确表达了TRAIL分子,并摸索了中试生产的条件,为其在肿瘤生物治疗中的应用奠定了基础。     

Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying α-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.     

A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.     

Synthesis and secretion of alpha-amylase by rice callus: evidence for differential gene expression

Rice seed callus expressed and secreted alpha-amylase at high levels. Twenty percent of the protein secreted by the callus was alphaamylase. The callus secreted about 840 mug alpha-amylase with 10.9 x 10(3) units of activity per gram dry weight callus per day. The alpha-amylase from callus exhibited a more complex isoform pattern than the germinating seed alpha-amylase. In addition, the level of mRNA expression by the five alpha-amylase gene groups was markedly different between callus and the germinating seed. The rice callus culture has features which it attractive as a potential system for expression proteins in plant cell fermentation systems.     

Functional solubilization of aggregation-prone TRAIL protein facilitated by coexpressing with protein isoaspartate methyltranferase

TRAIL was a tumor-specific protein in development as a novel anticancer therapeutic agent. Generally, when expressed in recombinant Escherichia coli, TRAIL protein was prone to form inclusion bodies. In this study, coexpression of human TRAIL protein and protein isoaspartate methyltranferase (PIMT) from E. coli on plasmid pBV–TRAIL–PCM in E. coli C600 was investigated to overcome the difficulties in soluble expression. The results showed that this PIMT coexpression strategy exerted a positive effect on the TRAIL protein expression in recombinant E. coli, which led to a mean increase in the intracellular concentration of soluble and total protein of TRAIL by 1.57-fold and 1.33-fold, respectively. At the same time, results also suggested that PIMT was a prospective partner for soluble expression of TRAIL protein.     

Current advances and expectations in tumor immunology]

Natural killer (NK) cells and Interferon (IFN)-gamma have been implicated in immune surveillance against tumor. We demonstrated the critical role of perforin in NK cell-mediated cytotoxic activity and anti-tumor effect in IFN-gamma inducible IL-12. And, we recently reported that TRAIL is constitutively expressed on a substantial proportion of murine NK cells in the liver, and which is responsible for spontaneous cytotoxicity and the anti-metastatic activity against TRAIL-sensitive tumor cells along with perforin and Fas ligand. Interestingly, the TRAIL expression on liver NK cells appeared to be regulated by endogenously produced IFN-gamma. Consisting with this finding, IL-12 and NKT cell specific ligand, alpha-Galactosylceramide (alpha-GalCer), induced TRAIL-mediated cytotoxcity and anti-tumor effect, and which was mediated by TRAIL expressed on IFN-gamma-activated NK cells. Tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein belonging to the TNF family, which preferentially induces apoptotic cell death in various tumor cells in vitro. Preclinical studies in mice and nonhuman primates have shown that administration of recombinant soluble forms of TRAIL could suppress the growth of TRAIL-sensitive tumor xenografts with no apparent systemic toxicity. These studies suggested a potential utility of TRAIL as a cancer therapeutic, although TRAIL expression at protein levels and its physiological roles in tumor surveillance has remained unknown. Presented findings provide the first evidence for the physiological function of TRAIL as a tumor suppressor.     

人类肿瘤坏死因子相关凋亡配体活性部位基因在毕赤酵母中的表达

探讨了人类肿瘤坏死因子相关凋亡配体 (TRAIL)生物活性部分在巴斯德毕赤酵母 (Pichiapastoris)中的分泌表达。由于TRAIL活性部位区第 149 15 0氨基酸含有一个Kex2位点 ,根据Pichiapastoris分泌表达的特点 ,对 149位氨基酸进行了改造 ,使其编码的氨基酸由Arg突变为Lys。这样使TRAIL在分泌的过程中不被Kex2切割 ,保证了片段的完整。序列分析正确后 ,将编码TRAIL的可溶区的基因片段插入到酵母表达载体pPIC9K中 ,使之位于α 因子信号肽下游 ,且与之同框 ,构建分泌型表达载体pPIC9K TRAIL。采用原生质体法将重组质粒转化Pichiapastoris菌株GS115 ,获基因工程菌株Pichiapastoris(pPIC9K TRAIL)。将工程菌用甲醇诱导培养 3～ 4d ,对摇瓶发酵的培养上清进行SDS PAGE、Westernblot分析和体外生物活性检测 ,结果发现发酵液中分子量约为 19kD和 38kD的蛋白质能被TRAIL多克隆抗体特异性识别 ,且具有在体外诱导肿瘤细胞凋亡的活性。通过薄层扫描分析显示目的蛋白最高可占可溶性总蛋白的 36 %。上述实验结果表明 ,在Pichiapastoris中表达的TRAIL能以寡聚体的形式存在并且具有生物学活性。     

Cost-effective large-scale expression of proteins for NMR studies

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and ¹³C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of ²H, ¹³C and ¹⁵N using auto-induction or isopropyl-β-d-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.