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.     

A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of beta-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for beta-galactosidase, C(12)FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular beta-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained beta-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular beta-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce beta-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between beta-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.     

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。     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.     

Predictive tool for recombinant protein production in Escherichia coli shake-flask cultures using an on-line monitoring system

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively.     

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。     

重组大肠杆菌合成左旋多巴条件的优化

通过对合成体系各组分对产物形成的影响的研究。分别求得了底物邻苯二酚,丙酮酸及乙酸氨的表观米氏常数,表观底物抑制常数和最适底物浓度,并得出合成左旋多巴的最适反应体系为：1%丙酮酸,1.2%邻苯二酚,2%乙酸铵,0.1?TA,0.2%亚硫酸钠,用氨水调pH至8.0。加入从与合成体系等体积培养液中收获的重组大肠杆菌细胞,于15℃反应16h,L-DOPA的产量为15.36g/L。     

Production of recombinant protein in Escherichia coli cultured in extract from waste product alga,Ulva lactuca

This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six‐months‐old were able to produce two‐fold more GFP protein than traditional Lysogeny Broth media. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:784–789, 2014     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH,a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin,is a promising therapeutic candidate for thromboembolic diseases.To develop SFH into a new thrombolytic agent,scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies.Here,we describe a pilot-scale cultivation and purification process for the production of SFH.A high-cell-density fed-batch cultivation for the production of SFH in E.coli was developed in a 40-L bioreactor,which produced about 1.1 g/L of recombinant protein.SFH was purified to homogeneity from the E.coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography,with over 99% purity and 54% recovery.Moreover,the residual endotoxin content was less than 0.5 EU/mL.The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay,respectively.     

产1，3-丙二醇重组大肠杆菌JM109（pEtac—dhaB—tac—yqhD）的构建

在生物柴油的生产过程中,最高可得到约10％的副产物甘油,副产物甘油的去向将成为生物柴油大规模产业化发展所面临的严峻问题。以生物柴油副产物甘油为原料耦合生产1,3-丙二醇,不仅解决了生物柴油副产物甘油的出路问题,同时降低了1,3-丙二醇的生产成本。本研究在前期工作的基础上,分别获得了来源于肺炎克雷伯氏茵的甘油脱水酶编码基因dhaB和来源于大肠杆菌的1,3-PD氧化还原酶同工酶编码基因yqhD,利用表达载体pEtac串联构建了重组质粒pEtac—dhaB—tac—yqhD,将其转化大肠杆菌得到产1,3-丙二醇重组大肠杆菌JM109（pEtac—dhaB-tac—yqhD）,降低了代谢中间产物3-羟基丙醛的积累,提高了1,3-丙二醇的产量。     

重组截短型人源结缔组织生长因子在大肠杆菌中的表达

用RT－PCR法从人胎盘组织中克隆出人源结缔组织生长因子（h-CTGF)cDNA序列867bp,将此cDNA亚克隆至表达载体pET-9a,重组质粒转化BL21（DE3）pLysS,诱导出N端缺失61个氨基酸残基的截短型rh－CTGF,表达量占总菌体蛋白的7％,主要以不溶性包涵体形式存在,采用离心、洗涤和凝胶过滤分离纯化后,行活性检测表明截短型rt-CTGF无刺激增殖活性,并对用原核表达rt-CTGF作了讨论,为制备抗体、CTGF表达调控和功能研究打下了基础。     

Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15

Abstract

Leishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1?g/L, 1.0?g/L, and 10?g/L for lactose and 20?μM, 100?μM, 500?μM, and 1000?μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100?μM (0.087?g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1?g/L (0.016?g/L). Thus, the induction with 100?μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration.     

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。     

Continuous bioconversion of n-octane to octanoic acid by recombinant Escherichia coli (alk(+)) growing in a two-liquid-phase Chemostat

Escherichia coli is able to grow on sugars in the presence of a bulk n-alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n-alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two-liquid-phase continuous cultures containing 5% (v/v) n-octane.In contrast to batch cultures of wild-tape E. coli grown in the presence of n-octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n-octane to n-octanoic acid by a recombinant E. coli (alk(+)) in a two-liquid-phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two-phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h(-1), reaching a continuous production rate of 0.5 g octanoate/L . h (4 tons/m(3) . year). (c) 1993 John Wiley & Sons, Inc.     

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018     

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli’s and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10¹ to 10⁷ copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.     

产普鲁兰酶重组大肠杆菌质粒稳定性的研究

通过对产普鲁兰酶的重组大肠杆菌E.coli BL21(DE3)/p ET28a-s-pul菌株在发酵过程中质粒稳定性和普鲁兰酶生成量的考察,发现不同宿主对质粒稳定性及酶活性有重要影响。本文利用E.coli BL21(DE3)p Lys S菌株为宿主,构建重组菌E.coli BL21(DE3)p Lys S/p ET28a-s-pul,通过控制外源蛋白的本底表达,提高了重组菌株的质粒稳定性。优化发酵培养基和发酵条件以后,重组菌产普鲁兰酶能力由480 U/m L提高至627 U/m L,增幅为30.6%。研究结果认为,严格控制外源蛋白的本底表达,是改善重组菌稳定性的重要方法之一。     

A kinetic model describing cell growth and production of highly active, recombinant ice nucleation protein in Escherichia coli

A structured kinetic model, which describes the production of the recombinant ice nucleation protein in different conditions, was applied. The model parameters were estimated based on the variation of the specific growth rate and the intracellular product concentration during cultivation. The equations employed relate the cellular plasmid content or plasmid copy number with the cloned-gene expression; these correlations were successfully tested on the experimental data. The optimal nutrient conditions for the growth of Escherichia coli expressing the inaZ gene of Pseudomonas syringae were determined for the production of active ice nucleation protein. The kinetics of the cultures expressing the inaZ gene were studied in a bioreactor at different growth temperatures and nutrient conditions.     

Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation

Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0.