Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production

Sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for recombinant protein production but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The efficacy of inducing single-cell suspension correlated positively with the increase in sulfation of the added polyanion. Unsulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension.Elimination of clumping in suspension culture by adding a dispersing agent can lead to enhanced recombinant protein production. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in a four-fold increase in volumetric yield of the recombinant glycosylated protein, human secreted alkaline phosphatase, and a two-fold increase in volumetric yield of the recombinant cytoplasmic protein, beta-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/mL (ca. 2.3 g/L) for beta-galactosidase under elevated oxygen have been obtained. The optimum volumetric yield of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen but unsupplemented medium is 6 to 11-fold higher than attached cultures, and 3-fold higher than the best yield obtained for SF21 cells in suspension at elevated oxygen and with nutrient supplementation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:191-205, 1997.     

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.     

Effects of autophagy inducers on recombinant antibody production in insect cells

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

     

Facile monitoring of avian reovirus σB expression and purification processes by tagged green fluorescent protein

Avian reovirus capsid protein σB was genetically fused with a histidine (His₆) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r² = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10⁶ cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

增殖细胞核抗原蛋白在Spodoptera frugiperda昆虫细胞中的表达及纯化

目的:利用昆虫细胞表达系统生产重组的人增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),并进行纯化和抗体结合特性鉴定。方法:以HeLa细胞逆转录的cDNA为模板,扩增人PCNA基因,并插入杆状病毒载体AcMNPV。利用昆虫细胞得到PCNA基因的重组杆状病毒。病毒感染细胞表达蛋白,联合镍柱亲和层析和离子交换层析获得高纯度的重组人PCNA蛋白。ELISA法测定抗体结合特异性。结果:以HeLa细胞cDNA为模板得到的基因序列同GenBank的人PCNA基因序列一致。草地贪夜蛾细胞(Spodoptera frugiperda,Sf9)表达重组人PCNA(recombinant human PCNA,rPCNA)的最佳感染值(MOI)和感染时间分别为0.05h和144h。rPCNA的产量高达110mg/L细胞,纯度95%。间接ELISA法检测抗体结合特性,rPCNA的敏感性和特异性分别为93.3%和85.0%。结论:建立了rPCNA的表达和纯化方法,获得了高效表达、高度抗体结合特异性的PCNA蛋白,该蛋白质能进一步开发为PCNA相关疾病的体外诊断试剂盒,具较大的应用价值。     

High-Level Synthesis and Fate of Acetylcholine in Baculovirus-Infected Cells: Characterization and Purification of Recombinant Rat Choline Acetyltransferase

Rat choline acetyltransferase (ChAT) has been expressed at a high level in Spodoptera frugiperda Sf9 cells using a baculovirus expression system. A cDNA containing the coding sequence for ChAT was inserted into the transfer vector pAcYM1 to yield the recombinant vector pAcYM1/ChAT. Sf9 cells were then coinfected with pAcYM1/ChAT and the wild-type Autographa californica virus. One recombinant virus particle, containing the cDNA for ChAT, was selected that expressed a protein of 68.5 kDa. Forty hours after infection of cells with the recombinant virus, the specific activity of ChAT in the cytosol was 190 nmol of acetylcholine/min/mg of protein, accounting for approximately 24% of the cell cytosolic proteins as being ChAT. The apparent Km values of the enzyme for choline and acetyl-CoA were 299 and 221 microM, respectively, whereas the respective Vmax values were 10.6 and 11.4 mumol of acetylcholine/min/mg of protein. In addition, analysis of the protein revealed that ChAT is phosphorylated in Sf9 cells. About 0.5 mg of ChAT was obtained from a one-step purification procedure starting with 10(8) infected Sf9 cells. Addition of choline to the incubation medium led to accumulation of high amounts of acetylcholine in the cytosol of the infected cells. The neurotransmitter was not released by Sf9 cells in response to membrane depolarization or on ionophore-mediated calcium entry. Some acetylcholine, which most likely originated from cell death inherent to viral infection, accumulated in the culture medium. The infected insect cells, which synthesize and store neurotransmitter, provide a new and convenient model for analyzing synaptic transmission at the molecular level.     

重组蛋白在中国仓鼠卵巢细胞中高效表达的影响因素

高效表达重组蛋白 ,对于生物制药意义重大。大多数药用蛋白是糖蛋白 ,中国仓鼠卵巢细胞 (Chinesehamsterovarycell,CHO)是目前重组糖基蛋白生产的首选体系。影响外源蛋白在CHO细胞中表达的因素很多 ,从CHO细胞表达体系、表达载体系统、外源基因、表达细胞株的加压扩增与筛选、细胞大规模培养等方面对CHO高效表达加以阐述 ,同时提出存在的问题和未来的发展方向。     

Sequence-based factors influencing the expression of heterologous genes in the yeast Pichia pastoris--A comparative view on 79 human genes

High yield expression of heterologous proteins is usually a matter of "trial and error". In the search of parameters with a major impact on expression, we have applied a comparative analysis to 79 different human cDNAs expressed in Pichia pastoris. The cDNAs were cloned in an expression vector for intracellular expression and recombinant protein expression was monitored in a standardized procedure and classified with respect to the expression level. Of all sequence-based parameters with a possible influence on the expression level, more than 10 were analysed. Three of those factors proved to have a statistically significant association with the expression level. Low abundance of AT-rich regions in the cDNA associates with a high expression level. A comparatively high isoelectric point of the recombinant protein associates with failure of expression and, finally, the occurrence of a protein homologue in yeast is associated with detectable protein expression. Interestingly, some often discussed factors like codon usage or GC content did not show a significant impact on protein yield. These results could provide a basis for a knowledge-oriented optimisation of gene sequences both to increase protein yields and to help target selection and the design of high-throughput expression approaches.     

High yield secretion of the sweet-tasting protein lysozyme from the yeast Pichia pastoris

Hen egg lysozyme (HEL) is one of the sweet-tasting proteins. To understand why lysozyme is sweet, the enzyme was synthesized at high yields by a recombinant method. The mature HEL gene was cloned from a Taq polymerase-amplified PCR product into the Pichia pastoris expression and secretion vector pPIC6alpha. This expression vector contains both the Saccharomyces cerevisiae pre-pro alpha-mating factor secretion signal and the blasticidin resistance gene (bsd) for selection of transformants in bacteria and yeast. Expression of HEL was carried out in fermenter cultures. Culture supernatants were concentrated by ultrafiltration and purified by CM-ion exchange chromatography. Approximately 400 mgL-1 of recombinant HEL was obtained. The high yield of recombinant lysozyme enabled us to perform a sensory analysis in humans. The purified recombinant lysozyme elicited as a sweet taste sensation as does the lysozyme purified directly from egg white, and showed full lytic activity against cells of Micrococcus luteus. These results demonstrate that the P. pastoris expression system with the blasticidin S selection system is useful in producing recombinant sweet-tasting protein in active form at a high yield.     

Soluble expression of recombinant human CD137 ligand in <Emphasis Type="Italic">Escherichia coli</Emphasis> by co-expression of chaperones

CD137 ligand (CD137L) is a member of the tumor-necrosis factor superfamily that binds CD137 to provide positive co-stimulatory signals for T cells activation. Co-stimulation through CD137/CD137L has become one of the promising approaches for cancer therapy. Previous reports have shown that CD137L expressed in Escherichia coli resulted in inclusion bodies or low yield. In this study, the effects of five different chaperone teams on the soluble expression of recombinant human CD137L protein were explored and analyzed. The poor expression of CD137L in the cytoplasm of E. coli was improved significantly by co-expression of chaperone GroES-GroEL-Tf. After dual induction and affinity chromatography, purified recombinant CD137L was obtained at a yield of 3 mg protein per liter with purity greater than 98% from original undetectable level. Additionally, the purified recombinant CD137L could bind CD137-positive cells in a dose-dependent manner, markedly promote the growth of activated mice T cells, and elevate the release of IL-2. The present work provides an effective system for soluble expression of functional human co-stimulatory molecule CD137L, which will facilitate the clinical developments of recombinant protein drugs.     

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT₃ receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix^TM agitation system was chosen to overcome aggregation problems of BHK cells in suspension. In the process, cells were first grown to a density of 10⁶ cells/ml, the medium was then exchanged with fresh medium and the culture was infected with the recombinant virus at an estimated multiplicity of infection of 30. 24 h post infection we measured an expression level of 3 million functional 5-HT₃ receptors per cell. For harvesting, the cells were pelleted by centrifugation. The receptor protein was purified in a single step (Hovius et al., 1998) by exploiting the hexa-His tag at minimal protein loss (51% yield). Experiments to optimise expression resulted in yields up to 8 million receptors per cell, when the pH of a suspension culture was controlled at pH 7.3. Rapid virus generation and protein production, high protein yields as well as successful large scale application have made the SFV expression system attractive to produce large quantities of recombinant protein in a very short time. After optimisation of the expression conditions (in particular by setting the pH at 7.3), yields were increased twofold.     

Recombinant protein excretion in Escherichia coli JM103[pUC8]: Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability

The presence of a high copy number plasmid (pUC8) was found to affect integrity of the cell envelope of Escherichia coli JM103, causing in turn significant release of the plasmid-encoded protein (beta-lactamase). The alterations in cell membrane permeability were evident from the increased susceptibility of recombinant cells to deoxycholic acid and methylene blue, which did not have appreciable effect on plasmid-free cells. The deteriorated cell membrane structure also resulted in a substantial reduction in specific growth rate and mass yield of plasmid-bearing cells. Further enhancement in beta-lactamase excretion was achieved by permeabilizing cell membrane with ethylenediaminetetraacetate (EDTA) and phenethyl alcohol (PEA). Unlike other commonly used physical and chemical methods for releasing the enzymes accumulated in the cells, application of EDTA and PEA at appropriate concentrations neither led to cell death nor interrupted synthesis of the plasmid-encoded protein. While in situ application of PEA was complicated due to interference with beta-lactamase activity, in situ application of EDTA was found to be an efficient way of releasing the recombinant protein without sacrificing its productivity. The experimental results demonstrate that the presence of EDTA and PEA can substantially reduce the growth rate differential between plasmid-free and plasmid-bearing cells, suggesting possible improvement of plasmid stability by application of these cell membrence-permeabilizing agents on a periodic basis.     

On-column refolding and purification of transglutaminase from Streptomyces fradiae expressed as inclusion bodies in Escherichia coli

Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture.     

Production of hantavirus Puumala nucleocapsid protein in Saccharomyces cerevisiae for vaccine and diagnostics

The production of hantavirus Puumala nucleocapsid (N) protein for potential applications as a vaccine and for diagnostic purposes was investigated with Saccharomyces cerevisiae as a recombinant host. The N protein gene and the hexahistidine tagged N (h-N) protein gene were expressed intracellular from a 2-microm plasmid vectors under the control of a fused galactose inducible GAL10-PYK promoter. For monitoring the recombinant gene expression, a h-N and a GFP fusion protein was used. Different cultivation strategies and growth media compositions were tested in shake flasks and a 5 l bioreactor. When using defined YNB growth medium, we found the biomass yield to be unsatisfactorily low. Higher concentrated YNB medium, promoted cell growth but showed a pronounced inhibitory effect on heterologous gene expression. This phenomenon could not be attributed to plasmid losses, as we could demonstrate high stability of the vector under the applied cultivation conditions. Supplementation of YNB medium with extracts of plant origin resulted in increased biomass yields with concomitant high expression levels of the recombinant gene. The modified medium was used for fed-batch cultivations where basic metabolic features as well as growth parameters were determined in addition to recombinant gene expression. The maximal volumetric yield of N protein was 316 mg l(-1), the respective yield of h-N protein was 284 mg l(-1). Our study provides a basis for large-scale production of hantavirus vaccines, which satisfies economic efficiency as well as biosafety regulations for human applications.     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

On the effect of transient expression of mutated elF2α and elF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock

There are a growing number of reports on the beneficial effects of subphysiological temperature in vitro culturing (27–35°C) of mammalian cells on recombinant protein yield. However, this effect is not conserved across cell lines and target products, and our understanding of the molecular mechanism(s) responsible for increased recombinant protein yield upon reduced temperature culturing of mammalian cells is poor. What is known is that mammalian cells respond to cold-shock by attenuating global cap-dependent translation. Here, we have investigated the hypothesis that the cap-dependent attenuation of mRNA translation upon cold-stress of in vitro-cultured mammalian cells can be prevented, or at least alleviated, by overexpressing mutant translation initiation factors in Chinese hamster ovary and HeLa cells. We have shown that the transient coexpression of either an eIF2αSer⁵¹→Ala⁵¹ mutant or an eIF4ESer²⁰⁹→Glu²⁰⁹ mutant with firefly luciferase affects luciferase expression levels in a cell line and temperature dependent manner. Further, regardless of the coexpression of initiation factors, transient reporter gene expression was enhanced at subphysiological temperatures (<37°C), suggesting that reduced temperature cultivation can be used to improve the yield of recombinant protein during transient expression. The implications of these results upon cell engineering strategies involving manipulation of the translational apparatus for the enhancement of recombinant protein synthesis upon cold-shock are discussed. Joint first authors who contributed equally to this work     

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.