Modeling and observer design for recombinant Escherichia coli strain

A mathematical model for recombinant bacteria which includes foreign protein production is developed. The experimental system consists of an Escherichia Coli strain and plasmid pIT34 containing genes for bioluminescence and production of a protein, β-galactosidase. This recombinant strain is constructed to facilitate on-line estimation and control in a complex bioprocess. Several batch experiments are designed and performed to validate the developed model. The design of a model structure, the identification of the model parameters and the estimation problem are three parts of a joint design problem. A nonlinear observer is designed and an experimental evaluation is performed on a batch fermentation process to estimate the substrate consumption.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Analysis of two-stage recombinant bacterial fermentations using a structured kinetic model

A structured kinetic model has been employed to analyze the performance of a two-stage continuous fermentation of a recombinant Escherichia coli. Separating the cell growth phase from the gene expression phase in two fermentors minimizes the growth rate difference between the recombinant cells and the plasmid-free cells in the first fermentor, thereby increasing the plasmid stability. The plasmid-harboring cells from the first fermentor are continuously fed into the second fermentor, in which the foreign protein synthesis is turned on by the addition of the inducer. Consequently, the recombinant cells experience an immediate reduction in growth rates as soon as they enter the second stage and then recover to synthesize the foreign protein. To analyze the fermentation performance contributed by these cells with different intracellular foreign protein levels and growth rates, a novel method for determining the residence time distribution of the growing cells in the second stage has been formulated. Combined with this method, the structured kinetic model for recombinant bacterial cells is used to predict the plasmid stability and foreign productivity at various operation conditions, such as induction strength and dilution rates. This model can provide us with thorough understanding of the characteristics of the two-stage fermentations, and is useful for the development of large scale continuous cultures of recombinant bacteria.     

用遗传算法进行重组大肠杆菌发酵动力学的分析

重组大肠杆菌在诱导表达人表皮生长因子的过程促使细菌的生长受到抑制,一部分重组菌丧失了分裂能力,但仍保持着一定的代谢活力,分离成为存活但不能培养的细菌,根据大肠杆菌在表达外源蛋白过程中细胞生理状态的不同将细菌分为三类,提出一个描述诱导表达过程中重组大肠杆菌分离、生长的动力学模型．应用遗传算法对不同底物浓度的细胞生长、分离和产物合成的动力学参数进行了有效地估计,避免了传统算法可能陷于局部最优的问题,模型计算结果与实验结果吻合良好．分离模型在初始糖浓为5-20g/L的范围内可以较好地描述发酵过程中细胞生长、分离和目标产物表达的过程并具有一定的预测能力．     

外源表位在Salmonella菌毛上的表达

过去的20年中,在细菌表面展示外源多肽的表达系统的研究取得了重要进展。而其中相当一部分是以细菌菌毛作为表达载体用于表达外源多肽或蛋白。本文将详述一种特殊的利用基因置换构建的沙门菌菌毛外源多肽展示系统,同时介绍一些其他的菌毛展示系统并探讨他们的优劣性。     

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.     

Development of a novel vector system for programmed cell lysis in Escherichia coli

Although widely used as a host for recombinant protein production, Escherichia coli is unsuitable for massive screening of recombinant clones, owing to its poor secretion of proteins. A vector system containing T4 holin and T7 lysozyme genes under the control of the ptsG promoter derivative that is inducible in the absence of glucose was developed for programmed cell lysis of E. coli. Because E. coli harboring the vector grows well in the presence of glucose, but is lysed upon glucose exhaustion, the activity of the foreign gene expressed in E. coli can be monitored easily without an additional step for cell disruption after the foreign gene is expressed sufficiently with an appropriate concentration of glucose. The effectiveness of the vector was demonstrated by efficient screening of the amylase gene from a Bacillus subtilis genomic library. This vector system is expected to provide a more efficient and economic screening ofbioactive products from DNA libraries in large quantities.     

Polyclonal antibodies against the human cell surface CD34 marker

A highly efficient and inexpensive laboratory method of production and purification of polyclonal antibodies against the human cell surface CD34 marker was developed. It was demonstrated that unglycosy-lated recombinant protein cloned in E. coli cells and containing the extracellular fragment of the human CD34 antigen maintained the necessary antigenic determinants during isolation from bacteria and during immunization, induced the production of specific polyclonal antibodies, which could recognize the native antigen on the cell surface. The obtained antibodies can be used for CD34⁺ cell phenotyping by the immunocytochemistry and flow cytometry methods.     

Use of a stress-minimisation paradigm in high cell density fed-batch Escherichia coli fermentations to optimise recombinant protein production

Production of recombinant proteins is an industrially important technique in the biopharmaceutical sector. Many recombinant proteins are problematic to generate in a soluble form in bacteria as they readily form insoluble inclusion bodies. Recombinant protein solubility can be enhanced by minimising stress imposed on bacteria through decreasing growth temperature and the rate of recombinant protein production. In this study, we determined whether these stress-minimisation techniques can be successfully applied to industrially relevant high cell density Escherichia coli fermentations generating a recombinant protein prone to forming inclusion bodies, CheY–GFP. Flow cytometry was used as a routine technique to rapidly determine bacterial productivity and physiology at the single cell level, enabling determination of culture heterogeneity. We show that stress minimisation can be applied to high cell density fermentations (up to a dry cell weight of >70 g L^?1) using semi-defined media and glucose or glycerol as carbon sources, and using early or late induction of recombinant protein production, to produce high yields (up to 6 g L^?1) of aggregation-prone recombinant protein in a soluble form. These results clearly demonstrate that stress minimisation is a viable option for the optimisation of high cell density industrial fermentations for the production of high yields of difficult-to-produce recombinant proteins, and present a workflow for the application of stress-minimisation techniques in a variety of fermentation protocols.     

Down-regulation of acetate pathway through antisense strategy in Escherichia coli: improved foreign protein production

A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale protein production processes.     

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.     

An automated home-built low-cost fermenter suitable for large-scale bacterial expression of proteins in Escherichia coli

We have developed an automated fermentation system for cost-efficient upscaling of protein expression in bacteria. The system, built for use by nonbiotechnologists, can be assembled mostly from standard laboratory equipment and allows a largely unattended growth of bacteria to OD 25 (at 600 nm) in a 12 L vessel. The typical yield of 250-350 g of wet weight cell pellet per run, which is equivalent to the biomass obtained from 250 shake flask cultures containing 400 mL Luria-Broth medium each, facilitates the production of large amounts of purified recombinant protein without the laborious need for optimization of expression and purification conditions.     

Foot and mouth disease virus polyepitope protein produced in bacteria and plants induces protective immunity in guinea pigs

The goal of this project was to develop an alternative foot and mouth disease (FMD) vaccine candidate based on a recombinant protein consisting of efficient viral epitopes. A recombinant gene was designed that encodes B-cell epitopes of proteins VP1 and VP4 and T-cell epitopes of proteins 2C and 3D. The polyepitope protein (H-PE) was produced in E. coli bacteria or in N. benthamiana plants using a phytovirus expression system. The methods of extraction and purification of H-PE proteins from bacteria and plants were developed. Immunization of guinea pigs with the purified H-PE proteins induced an efficient immune response against foot and mouth disease virus (FMDV) serotype O/Taiwan/99 and protection against the disease. The polyepitope protein H-PE can be used as a basis for developing a new recombinant vaccine against FMD.     

Quantification of proteomic and metabolic burdens predicts growth retardation and overflow metabolism in recombinant Escherichia coli

Escherichia coli has been the host organism most frequently investigated for efficient recombinant protein production. However, the production of a foreign protein in recombinant E. coli often leads to growth deterioration and elevated secretion of acetic acid. Such observed phenomena have been widely linked with cell stress responses and metabolic burdens originated particularly from the increased energy demand. In this study, flux balance analysis and dynamic flux balance analysis were applied to investigate the observed growth physiology of recombinant E. coli, incorporating the proteome allocation theory and an adjustable maintenance energy level (ATPM) to capture the proteomic and energetic burdens introduced by recombinant protein synthesis. Model predictions of biomass growth, substrate consumption, acetate excretion, and protein production with two different strains were in good agreement with the experimental data, indicating that the constraint on the available proteomic resource and the change in ATPM might be important contributors governing the growth physiology of recombinant strains. The modeling framework developed in this work, currently with several limitations to overcome, offers a starting point for the development of a practical, model-based tool to guide metabolic engineering decisions for boosting recombinant protein production.     

Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation

Addition of stimulatory chemical such as glycerol was found to increase recombinant protein production in Chinese hamster ovary (CHO) cells. However, glycerol influenced cell mitosis and reduced cell growth rate. We developed a controlled proliferation strategy to utilize the stimulation of glycerol on recombinant protein production and mitigate the problem of growth inhibition. The approach is to apply a two-stage process, where cells are cultured without glycerol for a period of time in order to obtain enough cell density and then glycerol is added to achieve high specific productivity. In addition, a model for predicting the profiles of cell proliferation and recombinant protein production was developed and validated. A two-stage process, addition of 1% glycerol after 1 day of growth, could increase the final production of macrophage-colony stimulating factor (M-CSF) by 38% compared with the value obtained without addition of glycerol.     

Secretion of the antibacterial recombinant protein enbocin

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.     

A new technique for producing recombinant baculovirus directly in silkworm larvae

A new technique for the direct production of recombinant baculovirus in the silkworm larvae is described. To assess the utility of this method, a combination of Bombyx mori nucleopolyhedroviral genome, transfer vector and Lipofectin was co-injected directly into newly ecdysed fifth instar, silkworm larvae. The recombinant virus was obtained from the hemolymph of injected larvae and the hemolymph then re-injected into the larvae as an inoculum. This resulted in a high-level production of foreign protein in the silkworm larvae. This technique produces easy and rapid recombinant protein production in silkworms.     

Bombyx mori protein disulfide isomerase enhances the production of nuecin, an antibacterial protein

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpressions of foreign proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.     

利用ELP自断裂标签在大肠杆菌中生产抗菌肽Oxysterlin 1

抗菌肽是目前最有希望的抗生素替代品,但是使用重组技术生产抗菌肽的策略大多步骤烦琐且价格昂贵,不利于抗菌肽的规模化生产。Oxysterlin 1是一种新型的天蚕素抗菌肽,主要对革兰氏阴性菌有抗菌活性,具有较低的细胞毒性。文中利用一种简单经济的方法在大肠杆菌中实现Oxysterlin 1的表达和纯化。将Oxysterlin 1基因克隆到含有弹性蛋白样多肽Elastin-like polypeptide (ELP) 和蛋白质内含肽 (Intein) 的载体中,构建重组表达质粒pET-ELP-I-Oxysterlin 1。重组蛋白在大肠杆菌中主要以可溶性形式表达,进而通过简单的盐析和pH改变便可对目标小肽进行纯化。最终得到的Oxysterlin 1的产量约为1.2 mg/L,抑菌试验显示出预期活性,为抗菌肽的规模化生产及深入研究其抑菌机理奠定基础。     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.