Prokaryotic expression,in vitro folding,and molecular pharmacological characterization of the neuropeptide Y receptor type 2

G protein‐coupled receptors (GPCRs) are a class of membrane proteins that represent a major target for pharmacological developments. However, there is still little knowledge about GPCR structure and dynamics since high‐level expression and characterization of active GPCRs in vitro is extremely complicated. Here, we describe the recombinant expression and functional folding of the human Y₂ receptor from inclusion bodies of E. coli cultures. Milligram protein quantities were produced using high density fermentation and isolated in a single step purification with a yield of over 20 mg/L culture. Extensive studies were carried out on in vitro refolding and stabilization of the isolated receptor in detergent solution. The specific binding of the ligand, the 36 residue neuropeptide Y (NPY), to the recombinant Y₂ receptors in micellar form was shown by several radioligand affinity assays. In competition experiments, an IC₅₀ value in low nanomolar range could be determined. Further, a K_D value of 1.9 nM was determined from a saturation assay, where NPY was titrated to the recombinant Y₂ receptors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Response dynamics of 26-, 34-, 39-, 54-, and 80-kDa proteases in induced cultures of recombinant Escherichia coli

Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are "shocked" by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. (c) 1993 John Wiley & Sons, Inc.     

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。     

Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.     

提高大肠杆菌分泌表达重组蛋白的研究进展

大肠杆菌是表达重组蛋白的常见宿主之一。重组蛋白分泌到周质空间或胞外培养基中较之在胞内以包含体形式表达有许多优势。主要讨论大肠杆菌Ⅰ、Ⅱ型分泌机制,并总结近年来在提高重组蛋白分泌表达的策略方面取得的进展。     

Model based calculation of substrate/inducer feed-rate profiles in fed-batch processes for recombinant protein production

A model-based feed-rate profile optimization problem is discussed for the fed-batch recombinant protein production. Two optimization procedures, an evolutionary programming technique and a simplified method using the dynamic programming concept, are discussed and compared. Modeling as well as experimental results are presented.     

大肠杆菌表达的病毒样颗粒疫苗

重组病毒样颗粒是病毒衣壳蛋白外源表达的重要形式,形态结构与天然病毒高度相似,位于纳米尺度的大小易于被免疫系统识别,可激发机体产生保护性免疫反应,且不含有病毒基因,因此,是一种理想的疫苗形式,也是基于结构进行疫苗设计的重要结构载体。目前已上市的乙型肝炎疫苗、人乳头瘤病毒疫苗和戊型肝炎疫苗等基因工程疫苗均采用病毒样颗粒形式。大肠杆菌表达系统被广泛用于基因工程药物的生产,具有安全性好、生产周期短、易于放大生产等优点,在病毒样颗粒疫苗应用上具有良好前景。本文综述了利用大肠杆菌研制戊型肝炎疫苗和人乳头瘤病毒疫苗的进展,特别是这些病毒样颗粒疫苗的表达及组装、表位结构特征和临床试验结果。     

内皮型氧化氮合酶FAD间区在大肠杆菌中的表达

为了研究内皮型氧化氮合酶（ｅＮＯＳ）的功能、功能调节以及结构与功能的关系．通过ＰＣＲ技术克隆出ｅＮＯＳＦＡＤ间区８０１～９０２ＡＡ肽段的编码基因,插入ｐＥＴ２８ａ（＋）表达质粒中构建成ｐＥＴ２８ａ／ｅＮＯＳ２重组表达质粒,经转染在大肠杆菌中成功表达．表达蛋白经金属离子螯合亲和层析和ＳＤＳＰＡＧＥ回收纯化,得蛋白质纯品．为ｅＮＯＳ特异性抑制肽的筛选和ｅＮＯＳ特异性抗体的制备提供了必要的准备     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

人精子膜蛋白片段在沙门氏菌中的表达——一种制备抗生育疫苗的途径(英文)

合成编码一种人精子膜蛋白YWK-Ⅱ胞外区的一段多肽片段的双链寡核昔酸链,HSD—2a。用平端连接的方法将其插入到沙门氏菌鞭毛基因fliC(d)的抗原表位IV高变区EcoRV位点,构建了重组质粒pLS408-H1。重组基因在鞭毛负性aroA基因缺失的无致病性沙门氏菌S.dublin SL5928疫苗菌株中表达。经ELISA、电镜免疫胶体金法检测,表明HSD-2a编码的多肽片段成功地在沙门氏菌鞭毛表面表达。融合鞭毛蛋白分子量约为60kD。Western-Blot法检测,提纯的融合鞭毛蛋白与YWK-Ⅱ小鼠抗血清只在60 kD处出现一条结合带。携带重组质粒pLS408—H1的沙门氏菌疫苗菌株极有可能成为抗生育疫苗。     

重组大肠杆菌的高密度发酵和甘油生产条件的初步研究

在摇瓶中进行重组大肠杆菌菌株BL21高密度发酵条件的研究,考察了葡萄糖浓度、盐离子浓度、温度、接种量、发酵时间等对该菌株生产甘油的影响。初步确定底物浓度为2.5%,盐离子浓度0.2%,温度为37℃,接种量为2%,经24h的摇瓶发酵,甘油产量最高达6.8g/L。在30L发酵罐实验中、按初步确定的优化条件发酵26h,甘油产量可达46.67g/L,是LB/葡萄糖培养基中甘油产量的2.06倍。     

重组大肠杆菌的分批补料培养方法

在重组大肠杆菌的培养过程中,存在着菌体的高浓度与外源蛋白的高表达这一矛盾,使得重组菌的比生长速率通常远远低于宿主菌,限制了基因工程菌由实验室规模向工业化规模的转变。要实现重组大肠杆菌的高密度培养,最常用和最有效的方法就是分批补料流加培养。     

重组大肠杆菌高密度培养研究进展

重组大肠杆菌细胞高密度培养(High cell-density cultivation,HCDC)是获得高外源蛋白产率的一种重要策略,影响重组大肠杆菌高密度培养的因素主要有以下几个方面:重组体的构建及其稳定性,培养基成分,培养方式,培养条件及培养过程中抑制性代谢产物的积累等。从以上几个方面对近期的研究进展进行了综述。     

Preparation of recombinant six-histidine-tagged human LECT2, a chemotactic protein to neutrophils, in Escherichia coli

LECT2 is a chemotactic protein to neutrophils. A recombinant six-histidine-tagged human LECT2, (His)₆-LECT2, was expressed in E. coli using a pET21a(+) vector. The (His)₆-LECT2 was purified from the soluble fraction in E. coli as a single band in sodium dodesyl sulfate/polyacrylamide gel electrophoresis using three steps of column chromatography with Ni²⁺-charged nitrilo-triacetic acid (Ni-NTA) agarose, DEAE-Sepharose, and CM-Sepharose. The purified (His)₆-LECT2 was yielded with 96 μg from the soluble fraction of 1,500 ml culture of E. coli. The circular dichroism spectrum of (His)₆-LECT2 showed the folded structure, which is rich in β-sheet structure and rare in α-helix. This revised version was published online in June 2006 with corrections to the Cover Date.     

我国D2-43病毒株PrM-E基因的重组SFV的制备及生物学鉴定

 为构建含PrM E基因的重组SFV病毒 ,将含我国登革 2型病毒PrM E基因的SFV表达载体和辅助载体DNA线性化后 ,进行体外转录 .然后将获得的 2种RNA转录物共转染BHK2 1细胞 ,以RT PCR法证明转染后的细胞培养上清中含有重组SFV .进一步将该重组病毒经蛋白酶激活后可使BHK2 1细胞产生细胞病变 ,并且以间接免疫荧光法在感染细胞内可检测到登革 2型病毒所特有的蛋白 .含我国登革 2型病毒PrM E基因的重组SFV的获得 ,为进一步观察该重组病毒的免疫原性奠定了基础 ,从而为登革新型疫苗的研制提供新途径     

重组大肠杆菌高密度发酵生产RGD-TRAIL的条件优化

本文以一株产RGD-TRAIL的重组大肠杆菌为研究对象,在10L发酵罐中考查了诱导温度、pH值、溶氧、流加葡萄糖对重组大肠杆菌生长和RGD-TRAIL蛋白表达的影响。结果表明:诱导温度25℃,pH值控制7.0,溶氧控制30%,以5g·L~(-1)·h~(-1)流速流加葡萄糖最有利于菌体生长和蛋白表达,菌体收率和RGD-TRAIL产量分别达到45.99g·L~(-1)和160.2mg·L~(-1)。