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.     

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.     

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.     

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.     

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.     

New tools for recombinant protein production in Escherichia coli: A 5‐year update

The production of proteins in sufficient amounts is key for their study or use as biotherapeutic agents. Escherichia coli is the host of choice for recombinant protein production given its fast growth, easy manipulation, and cost‐effectiveness. As such, its protein production capabilities are continuously being improved. Also, the associated tools (such as plasmids and cultivation conditions) are subject of ongoing research to optimize product yield. In this work, we review the latest advances in recombinant protein production in E. coli.     

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni²⁺ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more -helix and less -structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签（EDA标签、MBP标签和GST标签）以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

Connection between gene dosage and protein stability revealed by a high-yield production of recombinant proteins in an E. coli LexA1(Ind-) background

Bacterial production of a plasmid-encoded bacteriophage P22 tailspike protein shows different yield and impact on cell viability in RecA+ LexA+, RecA- LexA+ and RecA+ LexA1(Ind-) backgrounds. In a LexA1(Ind-) context, we have observed lesser toxicity and higher productivity than in the wild-type strain, in which the bacterial growth was inhibited after induction of recombinant gene expression. Also, a negative effect of the incubation temperature on the growth of producing cells was also detected. By exploring the molecular basis of these inhibitory events, we found a connection between the dosage of the recombinant gene and the proteolytic stability of the encoded protein. Under both genetic and environmental conditions favoring higher plasmid copy number and consequently increasing the synthesis rate of the recombinant protein, enhanced protein degradation was observed in parallel with an important growth inhibition. Altogether, the obtained data suggest the existence of a critical concentration of recombinant protein over which cell proteolysis is stimulated at rates not compatible with optimal physiological conditions for bacterial growth.     

Clinical manufacturing of recombinant human interleukin 15. I. Production cell line development and protein expression in E. coli with stop codon optimization

Interleukin 15 (IL-15) has shown remarkable biological properties of promoting NK- and T-cell activation and proliferation, as well as enhancing antitumor immunity of CD8(+) T cells in preclinical models. Here, we report the development of an E. coli cell line to express recombinant human Interleukin-15 (rhIL-15) for clinical manufacturing. Human IL-15 cDNA sequence was inserted into a pET28b plasmid and expressed in several E. coli BL21 strains. Through product quality comparisons among several E. coli strains, including E. coli BL21(DE3), BL21(DE3)pLysS, BLR(DE3)pLysS, and BL21-AI, E. coli BL21-AI was selected for clinical manufacturing. Expression optimization was carried out at shake flask and 20-L fermenter scales, and the product was expressed as inclusion bodies that were solubilized, refolded, and purified to yield active rhIL-15. Stop codons of the expression construct were further investigated after 15-20% of the purified rhIL-15 showed an extraneous peak corresponding to an extra tryptophan residue based on peptide mapping and mass spectrometry analysis. It was determined that the presence of an extra tryptophan was due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre). As a novel strategy, a simple method of demonstrating lack of tRNA suppressors in the production host cells was developed to validate the cells in this study. The E. coli BL21-AI cells containing the rhIL-15 coding sequence with a triplet stop codon TAATAATGA were banked for further clinical manufacturing.     

Tracking the acetate threshold using DO-transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media

DO-transient nutrient controllers use the dissolved oxygen signal to attempt acetate threshold tracking during fed-batch cultivation of recombinant E. coli. Here we apply DO-transient control to the production of Jembrana disease virus protein in complex Super Luria medium and compare performance against a high-limit pH-stat controller. For induction at medium cell density (harvest between 31 and 32.5 g dcw L) a total productivity of 0.27 g L h was achieved as compared to 0.24 g L h with the high-limit pH-stat. For induction at high cell density (harvest at 60 g dcw L), decreased productivity (0.12 g L h) was attributed to the effect of acetate accumulation on recombinant protein formation and a concomitant lowering of the critical growth rate. Our results suggest that complex media provides a difficult environment for the application of acetate threshold tracking DO-transient control because of difficulties in re-oxidizing acetate, and apparent localized production of acetate below the production threshold (as detected by the DO-transient controller as SPOUR(crit)). Configuring the DO-transient controller to avoid aggressive threshold probing is suggested as a means to improve performance and reduce acetate accumulation in complex media.     

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.     

Process optimization for large-scale production of TGF-α-PE40 in recombinant Escherichia coli: effect of medium composition and induction timing on protein expression

The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-α -PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L⁻¹ to 850 mg L⁻¹ by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L⁻¹, respectively. Received 26 August 1996/ Accepted in revised form 10 December 1996     

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.     

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     

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     

Increased phenotypic stability and ethanol tolerance of recombinant Escherichia coli KO11 when immobilized in continuous fluidized bed culture

The recombinant Escherichia coli B strain KO11, containing chromosomally-integrated genes for ethanol production, was developed for use in lignocellulose-to-ethanol bioconversion processes but suffers from instability in continuous culture and a low ethanol tolerance compared to yeast. Here we report the ability cell immobilization to improve its phenotypic stability and ethanol tolerance during continuous culture on a 50 g/L xylose feed. Experiments conducted in a vertical tubular fermentor operated as a liquid-fluidized bed with the cells immobilized on porous glass microspheres were compared to control experiments in the same reactor operated as a chemostat without the support particles. Without cell immobilization the ethanol yield fell sharply following start-up, declining to 60% of theoretical after only 8-9 days of continuous fermentation. While immobilizing the cells did not prevent this decline, it delayed its onset and slowed its rate. With immobilization, a stable high ethanol yield (>85%) was maintained for at least 10 days, thereafter declining slowly, but remaining above 70% even after up to 40 days of fermentation. The ethanol tolerance of E. coli KO11 cells was substantially increased by immobilization on the glass microspheres. In ethanol tolerance tests, immobilized cells released from the microspheres had survival rates 2.3- to 15-fold higher than those of free cells isolated from the same broth. Immobilization is concluded to be an effective means of increasing ethanol tolerance in E. coli KO11. While immobilization was only partially effective in combating its phenotypic instability, further improvements can be expected following optimization of the immobilization conditions.     

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production

Prokaryotic expression systems are widely used to produce many types of biologics because of their extreme conveniences and unmatchable cost. However, production of recombinant human ferritin light chain (rhFTL) protein is largely restrained because its expression in Escherichia coli tends to form inclusion bodies (IBs). In this study, a prokaryotic expression vector (FTL‐pBV220) harboring the rhFTL gene was constructed using a pBV220 plasmid. The tag‐free rhFTL was highly expressed and almost entirely converted to soluble form, and thus the rhFTL was successfully self‐assembled into uniform nanoparticles in E. coli. To establish a simplified downstream process, a precipitation procedure based on the optimized incubation temperature, pH condition, and ionic strength was developed to remove impurities from the crude lysate supernatant. The rhFTL retained in the clarified supernatant was subsequently purified in a single step using Capto Butyl column resulting in a considerable recovery and high purity. The purified rhFTL was characterized and verified by mass spectrometry and spectral and morphological analyses. The results revealed that rhFTL exhibited highly ordered and fairly compact structures and the spherical structures were preserved.     

Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli

Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.     

大肠杆菌细胞抽提物制备方案的简化与优化

无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。