Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.     

Optimization of recombinant clumping factor of Staphylococcus aureus expression in Escherichia coli XLI-BLUE

Experiments for optimization of the clfA gene expression in E. coli XLI-Blue cells were conducted. The reducing of the strain efficiency and the elimination of the plasmid during the cultivation of a producer have been shown. Several methods for raising the amount of plasmid-containing cells and increasing the yield of recombinant protein were proposed. Bacterial growth medium was selected to make this process more efficient. In such conditions after IPTG induction of the recombinant cells we have received high level of a soluble protein (about 30% of total cellular protein). The recombinant protein containing the His tag on N-terminus was purified on IDA-Sepharose column with high yield.     

Optimization of recombinant human nerve growth factor production in the psychrophilic Pseudoalteromonas haloplanktis

The optimization of production strategy is a very useful tool to attain high level of recombinant protein at a low cost. A promising biotechnological application of psychrophilic bacteria is their use as non-conventional host for the recombinant production of useful proteins. The lowering of the expression temperature can in fact facilitate the correct folding of heterologous proteins that accumulate in insoluble form as inclusion bodies when produced in Escherichia coli. An example of such "difficult" proteins is the human nerve growth factor (hNGF). The gene encoding the mature form of hNGF was expressed in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 at 4 degrees C. Western blotting experiments demonstrated that the protein was produced in soluble form and translocated in the periplasmic space. Furthermore, an analytical gel filtration chromatography confirmed that the recombinant protein was largely in dimeric form. For a more efficient recombinant rhNGF production, the influence of cultivation operational strategies and growth conditions (medium composition, temperature, specific growth rate) on biomass yield and recombinant protein production was investigated in batch and chemostat cultivations. The highest product yield of soluble rhNGF (7.5mg(NGF)g(dryweight)(-1)) has been achieved in batch culture at 4 degrees C on Schatz medium with addition of tryptone and vitamins.     

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies

A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.     

基因重组大肠杆菌表达血红素加氧酶-1及培养条件优化

【背景】血红素加氧酶-1 (HO-1)具有抗氧化应激、抗凋亡和抗纤维化等多种生理效应,有望成为一种新型药物应用于临床疾病的治疗。【目的】构建表达HO-1的基因重组大肠杆菌(Escherichiacoli),并优化其表达培养条件,实现HO-1高产率的表达。【方法】PCR法克隆集胞藻(Synechocystissp.)PCC6803的HO-1基因(ho1),构建重组质粒pET-28a-ho1,转化大肠杆菌BL21(DE3)菌株,单因素实验优化表达培养基的种类、诱导剂添加时间、诱导培养时间、诱导剂浓度和诱导培养温度。【结果】构建了表达HO-1的基因重组大肠杆菌BL21(DE3)/pET-28a-ho1菌株,用甘油(GY)培养基培养至菌体浓度OD_(600)约为0.8时,加入终浓度为0.1 mmol/L的IPTG诱导,30°C诱导培养6 h,HO-1的表达量最高,Ni-NTA柱分离纯化得到的HO-1收率占细胞总蛋白的10.9%。【结论】获得了可溶性表达HO-1的基因重组大肠杆菌及其较佳的培养条件,为进一步研究集胞藻来源的HO-1的酶学性质和应用奠定了基础。     

Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.     

Optimization of Escherichia coli cultivation methods for high yield neuropeptide Y receptor type 2 production

The recombinant expression of human G protein-coupled receptors usually yields low production levels using commonly available cultivation protocols. Here, we describe the development of a high yield production protocol for the human neuropeptide Y receptor type 2 (Y2R), which provides the determination of expression levels in a time, media composition, and process parameter dependent manner. Protein was produced by Escherichia coli in a defined medium composition suitable for isotopic labeling required for investigations by nuclear magnetic resonance spectroscopy. The Y2 receptor was fused to a C-terminal 8x histidine tag by means of the pET vector system for easy one-step purification via affinity chromatography, yielding a purity of 95-99% for every condition tested, which was determined by SDS-PAGE and Western blot analysis. The Y2 receptor was expressed as inclusion body aggregates in complex media and minimal media, using different carbon sources. We investigated the influences of media composition, temperature, pH, and set specific growth rate on cell behavior, biomass wet weight specific and culture volume specific amounts of the target protein, which had been identified by inclusion body preparation, solubilization, followed by purification and spectrometric determination of the protein concentration. The developed process control strategy led to very high reproducibility of cell growth and protein concentrations with a maximum yield of 800 μg purified Y2 receptor per gram wet biomass when glycerol was used as carbon source in the mineral salt medium composition (at 38 °C, pH 7.0, and a set specific growth rate of 0.14 g/(gh)). The maximum biomass specific amount of purified Y2 receptor enabled the production of 35 mg Y2R per liter culture medium at an optical density (600 nm) of 25.     

Fermentation conditions for high-level expression of the tac-promoter-controlled calf prochymosin cDNA in Escherichia coli HB101

Escherichia coli HB101 harboring an expression plasmid that bears the calf prochymosin gene controlled by the tac promoter was cultivated under different conditions in order to find an optimal fermentation arrangement that would lead to maximal prochymosin yield. Our results indicate that it is advantageous to use lactose in the double role of inducer and carbon/energy source when foreign gene expression is controlled by the tac promoter and the gene product is only moderately toxic owing to its accumulation in the form of an intracellular body. Glucose, on the other hand, may be used when expression should be repressed. Growth temperature substantially influenced the specific rate of prochymosin and beta-lactamase gene expression and the plasmid copy number. Three phases were distinguished in the time course of the fermentation on lactose: exponential growth practically without prochymosin synthesis, linear growth with prochymosin synthesis, and prochymosin synthesis without growth of biomass. The synthesis of prochymosin in the form of intracellular inclusion body was accompanied by the loss of respiratory activity of the cell and the loss of its ability to multiply. Sixteen hours cultivation at 37 degrees C in a complex medium with lactose as inducer and carbon/energy source resulted in up to 30% of the volume and 48% of the total protein of biomass being accumulated for as prochymosin inclusion bodies. The concentration of extractable enzymatically active chymosin in the culture reached 12 mg/L.     

Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis

Aims: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression–secretion system. Methods and Results: The esp gene was fused with the N‐terminal Sec‐dependent signal sequence of the B. choshinensis cell wall protein and a C‐terminal hexa‐histidine‐tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. Conclusions: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20‐ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1‐l culture). Significance and Impact of the Study: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.     

Engineering <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to produce feruloyl esterase for the release of ferulic acid from switchgrass

The Aspergillus niger feruloyl esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium with a yield of ~2 mg/l. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interaction chromatography. The specific activity was determined to be 8,200 U/μg (pH 6.5, 20°C, 3.5 mM 4-nitrophenyl ferulate). The protein had a correct N-terminal sequence of ASTQGISEDLY, indicating that the signal peptide was properly processed. The FAE exhibited an optimum pH of 6–7 and operated optimally at 50°C using ground switchgrass as the substrate. The yeast clone was demonstrated to catalyze the release of ferulic acid continuously from switchgrass in YNB medium at 30°C. This work represents the first report on engineering yeast for the breakdown of ferulic acid crosslink to facilitate consolidated bioprocessing.     

基因工程人α心钠素发酵研究

本研究采用的基因工程菌为酵母Y33：：YFD71－3,其基因型为α,his,1eu,ade,suc．摇瓶培养时心钠素的表达水平为l～2rag／L。在含有葡萄糖、YNB以及不同量腺嘌呤、组氨酸和亮氨酸的YG培养基中作摇瓶培养．当细胞的生长由腺嘌呤限制时,蛋白的分泌有明显增加·在YG培养基中加入5g／L的CAA后腺嘌呤成为限制性基质,培养基中腺嘌呤、YNB和亮氪酸用量对心钠素的表达有很大影响。在5L反应器中进行补料分批培养,流加葡萄糖、YNB、cAA、腺嘌呤、组氨酸和亮氨酸,心钠素的最高浓度达到24．8mg／L。     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar     

Mathematical Model of Growth and Heterologous Hantavirus Protein Production of the Recombinant Yeast Saccharomyces cerevisiae

A segregated mathematical model was developed for the analysis and interpretation of cultivation data of growth of the recombinant yeast Saccharomyces cerevisiae on multiple substrates (glucose, maltose, pyruvate, ethanol, acetate, and galactose). The model accounts for substrate consumption, plasmid stability, and production level of a model protein, a modified nucleocapsid protein of the Puumala virus. Recombinant nucleocapsid proteins from different Hantaviruses have previously been demonstrated as suitable antigens for diagnostics as well as for sero‐epidemiological studies. The model is based on a system of 10 nonlinear ordinary differential equations and accounts for the influence of various factors, e.g., selective pressure for enhancing plasmid stability by formaldehyde or the toxic effects of the intracellular accumulation of the heterologous protein on cell growth and product yield. The model allows the growth of two populations of cells to be simulated: plasmid‐bearing and plasmid‐free yeast cells, which have lost the plasmid during cultivation. Based on the model, sensitivity studies in respect to parameter changes were performed. These enabled, for example, the evaluation of the impact of an increase in the initial concentration of nutrients and growth factors (e.g., vitamins, microelements, etc.) on the biomass yield and the heterologous protein production level. As expected, the productivity of the heterologous protein in S. cerevisiae is closely correlated with plasmid stability. The 25 free model parameters, including the yield coefficients for different growth stages and dynamic constants, were estimated by nonlinear techniques, and the model was validated against a data set not used for parameter estimation. The simulation results were found to be in good agreement with the experimental data.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

大肠杆菌抗氟乙酸变株的选育及应用

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E.coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon…     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.