GST-SUMO-MT融合蛋白在大肠杆菌中的自诱导表达

旨在考察在大肠杆菌中自诱导表达GST-SUMO-MT融合蛋白的可行性,并对自诱导培养条件及培养基成分进行优化,以提高蛋白产量。采用摇瓶培养,利用单因素和正交实验对工程菌自诱导的培养条件(诱导时间、诱导温度)和培养基组分(蛋白胨、酵母粉、甘油、葡萄糖、乳糖)进行优化,检测菌体浓度、可溶性目的蛋白的表达量。结果表明,自诱导培养基碳氮源的最优配比为:2%蛋白胨、2%酵母粉、0.3%甘油、0.05%葡萄糖和0.3%乳糖。重组菌自诱导表达最优发酵条件为:采用两阶段温度控制,37℃培养3 h,25℃继续培养13 h。此时可溶性目的蛋白的表达量和菌体浓度分别为LB培养基(IPTG诱导)的2.2倍和2.3倍。     

人胰高血糖素样肽-1与人血清白蛋白融合蛋白在毕赤酵母中的表达

目的：在巴斯德毕赤酵母中表达有降糖活性的人胰高血糖素样肽-1（hGLP-1）突变体（2Gly-hGLP-1）与人血清白蛋白（HSA）的融合蛋白。方法：为将GLP-1氨基酸序列第2位的丙氨酸（Ala）定点突变为甘氨酸（Gly）,根据毕赤酵母偏爱密码子合成编码2Gly-hGLP-1的基因;采用重叠PCR法拼接2Gly-hGLP-1和HSA的基因,使得2Gly-hGLP-1的C端与HSA的N端通过甘氨酸五肽接头连接;将该融合基因插入表达载体pPIC9构建为重组载体pPIC9/2Gly-hGLP-1-HSA,电击转化至毕赤酵母GS115细胞,通过表型筛选和诱导表达实验获得高效表达菌株;工程菌在5L发酵罐中培养后,对发酵产物进行分离纯化和生物学活性分析。结果：融合蛋白在5L发酵罐中的表达量约为200mg/L,经纯化后纯度可达95%以上;小鼠糖耐量实验表明该融合蛋白具有明显的控血糖活性。结论：在毕赤酵母中分泌表达的融合蛋白2Gly-hGLP-1-HSA具有降血糖活性。     

人胰高血糖素样肽-1突变体基因的克隆及表达

目的：克隆人胰高血糖素样肽-1突变体（^2Gly-hGLP-1)基因,高效表达GST-^2Gly-hGLP-1融合蛋白.方法:在获得重组hGLP-1基因工程菌基础上,利用定点突变技术改造其第2位丙氨酸为甘氨酸,经酶切克隆于pGEM-7z( )载体中,构建pGEM-4T-3/^2Gly-hGLP-1融合表达规模.SDS-PAGE和凝胶扫描分析,融合蛋白以可溶形式存在,其表达量占菌体总蛋白的29.7%。表达产物经亲和层析纯化后纯度在95%以上,免疫印迹证实,该融合蛋白可被异性hGLP-1（7-37）抗体所识别。结论：为产业化规模制备hGLP-1突变体提供技术线路。     

利用GAP启动子在毕赤酵母中表达与纯化GLP-1类似物

胰高血糖素样肽-1(GLP-1)能提高II型糖尿病患者β胰腺细胞的胰岛素分泌量并能促进β胰腺细胞增殖,是潜在的糖尿病治疗药物。设计一种GLP-1类似物AGGH,即GLP-1(A2G)的二联体与人血清白蛋白(HSA)的N端连接,并在融合蛋白GGH前添加一个丙氨酸(A)。PCR获得融合基因aggh,将融合基因连接到p GAPZαA质粒中。在酵母中利用甘油醛三磷酸脱氢酶(GAP)启动子组成型表达外源蛋白AGGH。研究结果显示:筛选获得表达重组菌株,基因组PCR和western-blot验证正确;以葡萄糖为最优碳源培养下,表达量达到68 mg/L;5 L发酵罐中,发酵52 h蛋白产量最高达238 mg/L。蛋白经四步纯化后,获得纯度为95.8%的AGGH融合蛋白。与利用醇氧化酶1(AOX1)启动子表达的AGGH比较,发现两者产量和活性没有明显差异。但是,GAP启动子表达获得AGGH融合蛋白更加方便,且发酵时间减少了27.8%(20 h)。     

截短型胰高血糖素样肽-1的融合构建与表达

为了延长截短型胰高血糖素样肽1（shorted glucagon like peptide1,sGLP1）在血浆中的半衰期,将sGLP1的C端与人血清白蛋白（human albumin,HSA）的N端通过6个氨基酸的柔性连接子融合在一起,构建了融合表达菌株Pichia pastoris GS115/sGLP1HSA。以4g/L G418筛选出来的阳性工程菌株,经5L发酵罐培养,甲醇诱导48h,表达量可达0.8g/L。培养液经超滤、葡聚糖G25除盐、蓝色凝胶亲和层析纯化后,经HPLC分析产物纯度达95%,回收率达60%。正常小鼠糖耐量实验和对GKⅡ型糖尿病模型鼠的治疗证明sGLP1/HSA具有明显的降糖效果,为获得大量sGLP1/HSA融合蛋白进行临床实验研究奠定了基础。     

抗肿瘤肽QX3-1融合蛋白在大肠埃希菌中表达条件的优化

优化抗肿瘤肽QX3-1融合蛋白在大肠埃希菌中的表达条件,提高表达量。从培养基、诱导温度、诱导剂浓度和诱导时间4个单因素实验入手,优化QX3-1融合蛋白的表达条件,通过聚丙烯酰胺凝胶电泳(SDSPAGE)和Band Scan凝胶分析软件分析QX3-1融合蛋白的表达量。p ED-QX 3-1/BL21重组菌在LB、大豆肉汤、TB和酪蛋白胨(自制) 4种培养基中,于酪蛋白胨培养基中的表达量最高;在20、25和37℃三种诱导温度下,于37℃环境中表达量最高;在1、5、10、15和20 mmol/L乳糖浓度的诱导下,于10 mmol/L乳糖诱导最佳;在30 h的诱导时间内,于第24小时,融合蛋白的表达量最高。从培养基、诱导温度、诱导剂浓度和诱导时间4个变量中筛选出适合QX3-1融合蛋白表达的最佳组合,使融合蛋白的表达量达到了31. 2%,相比未优化前提高了近一倍。     

GLP-1-IgG4-Fc融合蛋白的表达与鉴定

将合成的人胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)突变体基因与IgG4抗体的Fc部分进行融合获得GLP-1-IgG4-Fc片段,获得的基因片段与pXC17.4载体进行连接,用电转化方法将线性化质粒稳定转染CHO-K1细胞,通过Clone Pix 2筛选出高表达细胞株,产量达1.5g/L。收集培养上清并经Protein A和Source 30Q纯化,得到的GLP-1-IgG4-Fc融合蛋白,SDS-PAGE纯度高于95%,高效液相色谱(high performance liquid chromatography,HPLC)纯度和毛细管区域凝胶电泳(capillary zone electrophoresis,CZE)纯度均不低于80%,尺寸排阻层析(size-exclusion chromatography,SEC)纯度高于99%。经质谱和肽图谱测定,分子量与理论值一致,肽图谱序列与对照品高度一致。生物学活性分析表明,GLP-1-IgG4-Fc融合蛋白具有促进表达有GLP-1受体的HEK293细胞分泌环磷酸腺苷(cyclic adenosine monophosphate,c AMP)的活性,并且该活性与对照品高度相似。     

碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

长效GLP-1-IgG2σ Fc融合蛋白的真核表达与生物活性鉴定

目的:在真核细胞中融合表达胰高血糖素样肽1(GLP-1),纯化后探究产物的生物学活性。方法:通过预留酶切位点将合成的GLP-1-Ig G2σFc融合蛋白表达序列连入表达载体质粒,用电转方法将线性化质粒稳定转染CHO-K1细胞,通过亚克隆筛选出蛋白高表达细胞株;收集高表达细胞株培养上清并初步纯化,采用口服葡萄糖耐量试验(OGTT)探究GLP-1融合蛋白降低血糖的生物学活性。结果:经过一轮亚克隆,筛选得到的细胞株的GLP-1融合蛋白产量达1 g/L,OGTT实验结果表明GLP-1融合蛋白具有降血糖生物活性。结论:GLP-1变体和Ig G2σ的Fc段连接是一种可行的融合蛋白形式,融合蛋白具有GLP-1受体激动剂的降糖作用,并且细胞株的筛选方式和初步纯化方法也具有可行性,为后续的体内、外活性研究和纯化方法确定提供了基础。     

重组人胰高血糖素样肽-1突变体的分离纯化及活性分析

对基因工程构建的含人胰高血糖素样肽1(hGLP1)突变体的工程菌株进行诱导表达,分离纯化N末端第二位突变的2GlyhGLP1突变体.IPTG诱导4h,收获的菌体经超声破碎后,裂解液用GlutathioneSepharose4B亲和层析纯化GST2GlyhGLP1融合蛋白,经CNBr裂解、SephadexG25柱脱盐、QAESepharoseFF阴离子交换柱层析和RPC18柱脱盐,得到纯度大于98%的重组2GlyhGLP1.Western印迹分析证实,该突变体可被特异性hGLP1抗体所识别.生物学活性分析表明,2GlyhGLP1具有明显的降血糖活性和促胰岛素分泌活性(P<0.001).     

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a promising type 2 anti-diabetic agent, glucagon-like peptide-1 (GLP-1) is attracting more and more interest. Mutated GLP-1 (mGLP-1) is an analog of native GLP-1. To facilitate the production and purification of mGLP-1, auto-induction and on-column cleavage was employed in this study. By using auto-induction system, after 24 h of shaking culture, about 12.6 g wet bacterial cells could be obtained from 1 l medium, and this was about 3.6 times more than that of the IPTG-induction group. After disruption and centrifugation, the fusion protein was directly purified and cleaved on Ni–Sepharose 6 Fast Flow column. Then, RESOURCE15 RPC column was used for further purification. By using these two steps of purification, about 1.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 g wet bacterial cells. In the bioactivity study, mGLP-1 displayed a significant and dose-dependent glucose-lowering activity. These results suggested that auto-induction and on-column cleavage could facilitate the production and purification of mGLP-1. These methods could also be applied to the preparation of other proteins and peptides.     

液化沙雷氏菌磷脂酶A1的克隆表达及乳糖自诱导发酵

为了利用大肠杆菌高效生产重组磷脂酶,克隆了液化沙雷氏菌磷脂酶A1的编码基因pla,分别使用pET-28a(+)和pET-20b(+)载体,实现了磷脂酶A1在大肠杆菌BL21(DE3)中的功能表达.重组菌利用载体pET-28a(+)在原始信号肽的介导下胞外PLA1酶活达40.8 U/mL,占总酶活的91％.重组菌转接至优化后的发酵诱导培养基:蛋白胨10 g/L,酵母粉5g/L,葡萄糖0.8 g/L,乳糖5 g/L,25 mmol/L Na2HPO4,25 mmol/L KH2PO4和1 mmol/L MgSO4;菌体生长6h后,添加7.5 g/L的甘氨酸,37℃恒温发酵24 h,重组菌胞外PLA1酶活达到128.7 U/mL.     

Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

Recombinant production of mGLP-1 by coupling of refolding and intein-mediated self-cleavage (CRIS)

Glucagon-like peptide-1 as an endogenous glucose-lowering peptide is a promising candidate for anti-diabetic drug development. Here, we developed a convenient method by coupling of refolding and intein-mediated self-cleavage (CRIS) to improve the recombinant production of a mutated glucagon-like peptide-1 (mGLP-1). Bacterial cell culture employing auto-induction was performed at 37?°C to avoid the intracellular self-cleavage of the intein fusion protein. The impacts of urea, pH, and temperature on the efficiency of CRIS were tested, and then, the optimized CRIS was established. Using the optimized method, we obtained the purified mGLP-1 with a yield of 3.41?mg peptide/g bacterial cells which was 5.6-fold higher than before. After that, using chromatography, peptide electrophoresis, and mass spectrometry, we determined the purity and molecular weight of the purified peptide and then confirmed its glucose-lowering activity by performing glucose tolerance test in mice. These results suggest that CRIS is a relatively simple and efficacious method for the recombinant production of mGLP-1, and as a general method, it can also be used for the recombinant preparation of some other proteins and peptides.     

Expression and purification of exendin-4, a GLP-1 receptor agonist, in Escherichia coli

Exendin-4 is a 39 amino acid peptide isolated from salivary secretions of Gila monster (Heloderma suspectum). It shows 53% sequence similarity to glucagon-like peptide-1 (GLP-1), which is evaluated for the regulation of plasma glucose in type 2 diabetes. Exendin-4 is a potent and long-acting agonist of GLP-1 receptor. In the present study, the exendin-4 gene obtained by PCR with an enterokinase site at N-terminus and a termination codon at C-terminus was expressed in Escherichia coli strain BL21 (DE3) harboring pET32a(+). The fusion protein was purified by chromatography on Ni-NTA-agarose column. Recombinant exendin-4 was obtained by enterokinase cleavage of the fusion protein and subsequent purification. The yield of recombinant exendin-4 was 3.15mg/10g bacteria. The obtained recombinant exendin-4 shows glucose-lowering action in vivo.     

Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.     

海洋枯草芽孢杆菌产纤溶酶的诱变育种与发酵工艺优化*

目的:对海洋来源的具有产纤溶酶能力的枯草芽孢杆菌(Bacillus subtilis)LC6-1进行紫外诱变,得到高产且稳定的突变株PW6-3,对该突变株发酵产酶的条件进行优化。方法:采用单因素和正交试验进行发酵培养基组分和培养条件的优化。结果:突变株PW6-3的酶活力为(6 960.21 ± 85.51)U/mL,较原始菌株提高了30.48%。以PW6-3为出发菌株,采用单因素及正交试验的方法对菌株进行发酵培养基组分与培养条件优化,最终得到的最佳培养基组分是:玉米淀粉30 g/L,玉米浆干粉40 g/L,CaCl₂ 3 g/L;最佳发酵培养条件是:32℃,转速200 r/min,接种量3%,pH 6.5,种龄18 h,发酵培养时间66 h,最终菌株的酶活力稳定在(9 203.63 ± 67.85)U/mL。结论:发酵工艺优化后,菌株PW6-3纤溶酶产量较诱变之前的菌株LC6-1提高72.53%,且发酵工艺成本较低,具有较好的经济效益。     

Citrus residues isolates improve astaxanthin production by Xanthophyllomyces dendrorhous

The wild strain and two astaxanthin-overproducing mutant strains, W618 and GNG274, of Xanthophyllomyces dendrorhous were analyzed in order to assess their ability to grow and synthesize astaxanthin in a minimal medium containing (per liter): 2 g KH2PO4, 0.5 g MgSO4, 2 g KNO3, and 1 g yeast extract, and supplemented with citrus residues isolates as a carbon source (citrus medium). The selected strain W618 was evaluated under various contents of citrus juice. At the content of 20% (v/v), the highest astaxanthin production reached 22.63 mg L(-1), which was two-fold more than that observed in yeast malt medium. Addition of 8% (v/v) n-hexadecane to the citrus medium was found to be optimal, increasing the astaxanthin yield by 21.7%.     

Optimization of fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K(2)HPO(4) 0.1%, and MgSO(4) 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25 degrees C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.