A highly efficient method for liquid and solid cultivation of the anaerobic hyperthermophilic eubacterium Thermotoga maritima

An efficient and economical medium--Thermotoga maritima basal medium (TMB)--was designed for the cultivation of T. maritima under either liquid or solid conditions. When the broth was flushed with N2 or CO2 throughout cell growth in a 10-L fermentor (pH controlled to 6.5), the maximum cell density (OD600) on TMB containing 1% glucose rose to 2.0 or higher (1.63 x 10(9) cells mL(-1)). Sheath-less cells observed by electron microscopy were captured during growth in the fermentor. Using a two-layer plating method, isolated single-well colonies were consistently obtained within 24 h on the TMB in modified tissue culture flasks. The minimal inhibitory chloramphenicol concentrations for T. maritima on TMB agar were 5 microg mL(-1) after 24 h and 48 h, and 25 microg mL(-1) at 72 h.     

Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride

Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD(600), which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17 mg per liter per OD(600) in strain BL21(DE3)pLysS by inclusion of 10-100mM magnesium chloride (MgCl(2)) in the culture medium. This improved by about five times the yields of fully active 54ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.     

The quiescent-cell expression system for protein synthesis in Escherichia coli.

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.     

The Quiescent-Cell Expression System for Protein Synthesis in Escherichia coli

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.     

Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli

Overexpression of rhIFN-alpha2b was obtained by synthesizing a codon optimized gene for IFN-alpha2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNalpha, which had the IFN-alpha2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible lambdaP(L) promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2g/L at a final OD(600) of 67. At this point, the IBs represented approximately 40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of approximately 3g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of approximately 3 x 10(9)IU/mg protein.     

毕赤酵母表达重组人白细胞介素11的连续培养研究

对毕赤酵母表达重组人白细胞介素11(rhIL11)工程菌的连续培养进行了工艺研究。连续培养在10L工作体积的发酵罐中进行,整个发酵过程历时约20天,发酵上清中rhIL11表达浓度约400mgml。在稀释率D=005h下,菌浓OD600达到100以上。     

50L规模中试发酵重组核苷二磷酸激酶A工程菌

中试规模发酵重组人核苷二磷酸激酶A（rhNDPK-A）工程菌,并对表达产物进行纯化。摇瓶培养一级种子至合适密度,以10%比例接种二级种子培养基,在7L发酵罐中培养至OD600为9.6～10.5,然后转入80L发酵罐中进行补料分批培养,所得菌体裂解后,经离子交换层析和亲和层析两步纯化得重组蛋白制品。结果表明,50L培养液经过10h培养后,湿菌收量为1560 g/批,NDPK-A表达量为23.8%。另外,补料方式对发酵密度有明显影响。与单纯补加碳源相比,同时补加碳源和氮源可以显著提高菌体产量,但对目的蛋白表达量地提高不明显。在较优条件下,菌体产量为(2220.00±169.71) g/批,蛋白表达量为(22.00±0.42) %,纯化后重组蛋白得率为510mg/L。产物可溶、密度适中、工艺简便的中试发酵条件的建立为高得率、大规模制备重组rhNDPK-A奠定了基础。     

Distinct human prolactin (hPRL) and growth hormone (hGH) behavior under bacteriophage lambda PL promoter control: temperature plays a major role in protein yields

When producing recombinant protein for therapy, it is desirable not only to obtain substantial amounts of the protein, but also to make sure that potential contaminants such as inducing agents are not present in the final product. To prevent this, one can use expression systems in which the promoter (lambdaP(L)) is activated by a temperature shift that denatures a repressor (e.g., cIts). In this manner, hGH was successfully expressed and secreted in Escherichia coli periplasm, with specific yields well above 1 microg ml(-1) A(600)(-1), after a temperature shift from 30 to 42 degrees C. However, attempts to express a related hormone, human prolactin, employing the same protocol were unsuccessful, providing 0.03 microg ml(-1) A(600)(-1) at the most. A process is described in which this labile protein is obtained from a cIts(-) strain under optimized temperature condition (37 degrees C). The highest periplasmic secretions of prolactin ever reported were thus obtained: 0.92+/-0.10 microg ml(-1) A(600)(-1) at an optical density of approximately 3 A(600) units in shake flask cultures and approximately 1 microg ml(-1) A(600)(-1), at an OD of 35 A(600) units, via a rapid and flexible batch feed process in laboratory bioreactor. Purified hPRL was monomeric, correctly processed (Mr=22,906), properly folded and bioactive (51.5+/-24.1 IU mg(-1)).     

Uptake and Metabolism of Serotonin by Ependymal Primary Cultures

Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system.     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

重组毕赤酵母发酵牛肉风味肽的中试研究

研究分泌表达16拷贝牛肉风味肽（beefy meaty peptide, BMP）毕赤酵母工程菌株(Pichia pastoris GS115-16B2)的500L发酵罐中试发酵工艺。在500L发酵罐中采用三步发酵法进行了3批次中试发酵实验,设定菌体培养阶段温度为30℃,pH5.0,诱导表达阶段温度为28℃,pH3.0。在发酵程中通过调节搅拌速度、通气量和罐压等措施来使DO维持在20%~35%左右,总共发酵126h（诱导时间为96h）,当诱导88h后（发酵118h）,600nm波长下的光密度值(OD600)达到184.5,菌体湿重达到318.7g/L,目的产物BMP的表达量达到最高为172 mg/L,实现了BMP的高效表达。通过中试发酵初步建立了工程菌P. pastoris GS115-16B2的中试发酵工艺,为BMP的进一步研究开发奠定了良好基础。     

Transmembrane chloride flux in tissue-cultured chick heart cells

To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)- 1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min- 1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent.     

Laccase-induced C–N coupling of substituted <Emphasis Type="Italic">p</Emphasis>-hydroquinones with <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid in comparison with known chemical routes

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD565) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.     

Biodegradation of gasoline by gellan gum-encapsulated bacterial cells

Encapsulated cell bioaugmentation is a novel alternative solution to in situ bioremediation of contaminated aquifers. This study was conducted to evaluate the feasibility of such a remediation strategy based on the performance of encapsulated cells in the biodegradation of gasoline, a major groundwater contaminant. An enriched bacterial consortium, isolated from a gasoline-polluted site, was encapsulated in gellan gum microbeads (16-53 microm diameter). The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non-encapsulated) cells. Encapsulated cells (2.6 mg(cells) x g(-1) bead) degraded over 90% gasoline hydrocarbons (initial concentration 50-600 mg x L(-1)) within 5-10 days at 10 degrees C. Equivalent levels of free cells removed comparable amounts of gasoline (initial concentration 50-400 mg x L(-1)) within the same period but required up to 30 days to degrade the highest level of gasoline tested (600 mg x L(-1)). Free cells exhibited a lag phase in biodegradation, which increased from 1 to 5 days with an increase in gasoline concentration (200-600 x mg L(-1)). Encapsulation provided cells with a protective barrier against toxic hydrocarbons, eliminating the adaptation period required by free cells. The reduction of encapsulated cell mass loading from 2.6 to 1.0 mg(cells) x g(-1) bead caused a substantial decrease in the extent of biodegradation within a 30-day incubation period. Encapsulated cells dispersed within the porous soil matrix of saturated soil microcosms demonstrated a reduced performance in the removal of gasoline (initial concentrations of 400 and 600 mg x L(-1)), removing 30-50% gasoline hydrocarbons compared to 40-60% by free cells within 21 days of incubation. The results of this study suggest that gellan gum-encapsulated bacterial cells have the potential to be used for biodegradation of gasoline hydrocarbons in aqueous systems.     

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.     

Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation

Previously reported techniques for the electrotransfer of foreign DNA into pediococci yield only a small number of transformants/mug DNA, especially when using undomesticated strains. This study reports an improved protocol for the electrotransformation of pediococci, based on trials using Pediococcus acidilactici P60 and the plasmid pRS4C1. The improved protocol yields from 2 to 3 log units more transformants than the previously reported methods, with up to (9.1+/-1.3)x10(4) transformants/mug of foreign DNA under the best conditions identified. The most important modifications proposed are an increase in electric field strength during electroporation (from 12.5 to 20kV/cm) and a reduction in lysozyme concentration during the preparation of electrocompetent cells (from 4000 to 2000U/ml): together, these two modifications greatly improve transformant yield. In addition, increasing cell culture time (from OD(600nm)=0.6 to OD(600nm)=1.0-1.2) and increasing dl-threonine concentration in the growth medium (from 20 to 40mM) also contribute to improved electrotransformation efficiency.     

High-level production of human leptin by fed-batch cultivation of recombinant Escherichia coli and its purification.

Human leptin is a 16-kDa (146-amino-acid) protein that is secreted from adipocytes and influences body weight homeostasis. In order to obtain high-level production of leptin, the human obese gene coding for leptin was expressed in Escherichia coli BL21(DE3) under the strong inducible T7 promoter. The recombinant leptin was produced as inclusion bodies in E. coli, and the recombinant leptin content was as high as 54% of the total protein content. For production of recombinant human leptin in large amounts, pH-stat fed-batch cultures were grown. Expression of leptin was induced at three different cell optical densities at 600 nm (OD600), 30, 90, and 140. When cells were induced at an OD600 of 90, the amount of leptin produced was 9.7 g/liter (37% of the total protein). After simple purification steps consisting of inclusion body isolation, denaturation and refolding, and anion-exchange chromatography, 144.9 mg of leptin that was more than 90% pure was obtained from a 50-ml culture, and the recovery yield was 41.1%.     

重组戊型肝炎病毒衣壳蛋白工程菌的高密度培养

在10L发酵罐中对戊型肝炎病毒衣壳蛋白在重组大肠杆菌中表达发酵工艺进行了研究,用分批培养方法探讨了不同培养基、培养基中磷酸盐浓度和Mg^2＋浓度等因素对菌体生长与重组蛋白表达的影响;用分批补料培养研究了不同的补料工艺对菌体生长与重组蛋白表达的影响,同时对重组菌诱导时期、诱导持续时间以及不同诱导温度表达包含体在尿素溶液中的溶解性进行了研究。结果表明,在优化后的培养基中,磷酸盐浓度、Mg^2＋浓度分别为80mmol/L 与20mmol/L时菌体生长与表达效果较好;分批补料培养中,37℃培养9h菌体达到对数期中期(约45OD600)为适宜诱导时期,加入终浓度为10mmol/L IPTG后诱导5h,OD600达到80以上,重组蛋白表达量达到29.74％,为最适收获菌体时间;37℃表达的包含体80％以上溶解在4mol/L的尿素溶液中,最终浓度达到14mg/mL; 10L发酵罐中确定的发酵工艺参数在30L发酵罐中进行了放大培养,10L发酵罐中确定的发酵工艺参数在30L发酵罐上具有可放大性与重复性, 可以应用于工业生产。     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.