Improvement of product yields by temperature-shifting of Escherichia coli cultures containing plasmid pOU140

Temperature shifting was investigated as a means of improving cloned-gene product yields form a recombinant Escherichia coli containing the temperature-sensitive plasmid, pOU 140. In a series of shaker flask fermentations recombinant cells were thermally induced for different time periods. The growth, stability, and plasmid product levels were followed, and the results indicate the existence of an induction time period that maximizes product yield. A sustained thermal induction results in recombinant cell death and instability, while exposure to a runaway temperature for minimal time periods does not give sufficiently high product yields. At intermediate cycling times, however, the recombinant cells remain stable, and the plasmid replication region is activated, resulting in higher product yields.     

Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli

Lycopene is produced by recombinant Escherichia coli expressing genes to encode for the lycopene biosynthesis. However, the productivity of lycopene seemed to be limited by many factors including product toxicity. In the present study, we have investigated physiology of recombinant E. coli during biosynthesis and in situ recovery of lycopene based on an organic/aqueous two-phase system. Lycopene, the 40-carbon molecule product, was little extracted from recombinant E. coli cells to octane or decane phase. However, partial digestion of cell walls with lysozyme promoted extraction of lycopene into the organic phases. Engineering of an organic/aqueous two-phase system allowed recombinant E. coli cells to produce ca. 40% larger amount of lycopene compared to that in a conventional aqueous single-phase system. Optimization of the in situ product recovery process will lead to further increase of product concentration and productivity.     

Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli

The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.     

结核分枝杆菌Rv3369基因的表达和纯化

在大肠杆菌中高效表达结核分枝杆菌Rv3369蛋白,获得纯化的重组蛋白rRv3369。通过聚合酶链反应(Polymerase chain reaction,PCR)扩增Rv3369基因;以质粒pET28a为表达载体,构建重组质粒,转化大肠杆菌BL21(DE3);以异丙基硫代半乳糖苷(IPTG)诱导表达目的蛋白,通过SDS-PAGE鉴定rRv3369在大肠杆菌中的表达,确定rRv3369在大肠杆菌中的表达形式;采用Ni-NTA His.Bind Resin来纯化重组蛋白。重组质粒pET28a-Rv3369中目的基因测序结果与报道序列相同。分子量约19.5kDa,表达量约占菌体总蛋白的18%,纯化后的重组蛋白样品经SDS-PAGE和激光密度扫描分析表明其纯度为90%以上,每100mL培养菌可获得1.56mg左右的重组蛋白。用亲和层析法纯化的重组蛋白纯度较好。     

A comparison of mathematical model predictions to experimental measurements for growth and recombinant protein production in induced cultures of Escherichia coli

Recombinant cell growth and protein synthesis by a recombinant Escherichia coli under various inducing conditions are compared to the predictions of a mathematical model. The mathematical model used was a combination of two literature models: (1) an empirical kinetic model for recombinant growth and product formation and (2) a genetically structured model of the lac promoter-operator on a multicopy plasmid. The experimental system utilized was recombinant E. coli CSH22 bearing the temperature-sensitive plasmid pVH106/172, which codes for the synthesis of beta-galactosidase and the other lac operon genes under the control of a lac promoter. Mathematical model predictions for recombinant beta-galactosidase yield and specific growth rate were compared with fermentation measurements of these same quantities for conditions of chemical induction with cyclic AMP and IPTG, copy number amplification (by shifting culture temperature), and combined chemical induction and copy number amplification. The model successfully predicted experimental product yields for most cases of chemical induction even though the product yields varied from 0.34 x 10(3) to 1500 x 10(3) units/g cell mass. The kinetic model also correctly predicted a decline in the specific growth rate with increasing levels of plasmid and recombinant protein. The model was less successful at predicting product amplification at high copy numbers. A comparison of model predictions and experimental results was also used to investigate some of the assumptions used in constructing the mathematical models.     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

Effects of plasmid amplification and recombinant gene expression on the growth kinetics of recombinant E. coli

An experimental study was undertaken to identify and quantitate the effects of plasmid amplification and recombinant gene expression on Escherichia coli growth kinetics. Identification of these effects was possible because recombinant gene expression and plasmid copy number were controlled by different mechanisms on plasmid pVH106/172. Recombinant gene expression of the lactose operon structural genes was under the control of the lac promoter and was activated by the addition of the chemicals, IPTG and cyclic AMP, to the fermentation medium. Plasmid content was amplified in a separate fermentation by increasing culture temperature since the plasmid replicon was temperature-sensitive. A final fermentation was performed in which both plasmid content and recombinant gene expression were induced simultaneously by adding chemicals and raising the culture temperature. Recombinant growth rates were found to be reduced by the expression of high levels of recombinant lac proteins in the chemical induction experiments and by the amplification of plasmid levels in the temperature induction experiment. High expression of recombinant lac proteins following chemical induction was accompanied by a loss in recombinant cell viability. In the plasmid amplification experiment, the recombinant cells did not lose viability but the recombinant product yields were much lower than those achieved in the chemical induction experiments. Combining temperature and chemical induction increased the recombinant product yield by a factor of 4400 but also lowered cellular growth rates by 70%.     

Fast isolation of recombinant baculovirus by antibody screening

We describe a rapid and efficient scheme for the isolation and purification of recombinant baculoviruses. The method is based on the detection of foreign proteins in cellular lysates of baculovirus-infected insect cells by antibody screening. The recombinant virus is purified by repeated serial dilutions. The method allows the identification and purification of recombinant viruses within 2 to 3 wk. This procedure selects for recombinant baculoviruses that highly overproduce the desired protein product.     

Analysis of glycoprotein heterogeneity by capillary electrophoresis and mass spectrometry

Glycosylation is a complex posttranslational modification that can result in extensive heterogeneity for recombinant glycoproteins produced by eukaryotic systems. The carbohydrate moiety of a recombinant glycoprotein may affect the immunogenicity, half-life, bioactivity, and stability of a potential therapeutic product. Regulatory authorities such as the US Food and Drug Administration demand increasingly sophisticated carbohydrate analysis to ensure product characterization, batch-to-batch consistency, and stability. The advent of new technologies for analysis of biopolymers by capillary electrophoresis and mass spectrometry has revolutionized strategies for recombinant protein characterization. In particular, recent advances in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry now permit relatively rapid and detaned assessment of glycoprotein and oligosaccharide structure. In this article, we describe some applications of capillary electrophoresis and mass spectrometry to monitor the glycosylation associated with a model recombinant glycoprotein, human interferon-γ.     

A亚群禽白血病病毒囊膜基因gp85片段在大肠杆菌中的表达

将RAV－1囊膜基因gp85片段来克隆到表达质粒pET-21d( )中得到重组表达质粒pET-21d-RAV-1env(BelⅡ/SalⅠ）序列分析表明该插入片段的核苷酸序列和阅读框都与RAV－1囊膜基因相应序列相同。用其转化大肠杆菌BL21（DE）3并经IPTG诱导,SDS－PAGE分析表明RAV－1囊膜基因融合蛋白表达产物约20kD,与理论值相符;IPTG诱导起始时间比诱持续时间对表达量的影响更大。     

Secretion of human interferon alpha 2b by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biologically active human interferon alpha 2b (HuIFNalpha-2b) was secreted into the culture medium by Streptomyces lividans transformed with recombinant plasmids coding for HuIFNalpha-2b fused to the Streptomyces exfoliatus M11 lipase A signal sequence. Levels were low, 15 or 100 ng/ml, depending on the plasmid used. Neither processed nor unprocessed HuIFNalpha-2b was detected in cell lysates of the transformants secreting the recombinant product. However, the secreted recombinant product was found to partially degrade when cultures reached the stationary phase by the action of an, as yet, unidentified mycelium-associated factor. Experimental evidence suggests that the degrading factor is related to mycelium-associated proteolytic activity.     

结核杆菌DnaA蛋白在耻垢分枝杆菌中的同源表达

目的：在耻垢分枝杆菌中表达重组结核杆菌DnaA蛋白并对表达产物进行鉴定。方法：用PCR的方法扩增结核杆菌dnaA基因并克隆至表达载体pMF406中,构建重组大肠杆菌-分枝杆菌穿梭质粒pMF-dnaA。经双酶切及测序鉴定后,用电转化的方法将重组质粒转至耻垢分枝杆菌mc2155中。用0.02%乙酰胺诱导重组耻垢分枝杆菌,对表达产物进行SDS-PAGE和Western blotting检测和鉴定。结果：重组耻垢分枝杆菌构建成功,SDS-PAGE及Western blotting结果显示该重组耻垢杆菌可以实现结核杆菌DnaA蛋白的同源高效表达。结论：结核杆菌DnaA蛋白的同源表达为结核杆菌DNA复制机制的研究奠定了基础。     

Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems

A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining.The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant.Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.Abbreviations 2-DE two-dimensional electrophoresis - rCHO recombinant Chinese hamster ovary cell - EPO erythropoietin - FBR fluidized bed reactor - IEF isoelectric focusing - IPG immobilized pH gradient - pI isoelectric point - SDS sodium dodecylsulfate - STR stirred tank reactor     

Autocatalytic replication of a recombinant RNA

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.     

Plant-made pharmaceuticals: leading products and production platforms

The number of approaches to recombinant protein production in plants is greater than ever before. Development of these new and improved technologies as production platforms for plant-made pharmaceuticals has and will continue to create new commercial opportunities in the pharmaceutical sector. However, it is inevitable that no single system will be optimal for the production of all recombinant proteins of interest in plants due to both the physical characteristics and the envisaged therapeutic application of each product. Here, we review a range of promising product/platform pairs emphasizing synergies during production and in clinical trials.     

Purification and characterization of an endo-polygalacturonase. From Aspergillus awamori

An extracellular endo-polygalacturonase (PGase) produced by Aspergillus awamori IFO 4033 was isolated from the culture filtrate. The enzyme was purified to a homogeneous preparation with cation-exchange and size-exclusion chromatographies. Its properties were investigated, comparing them with that of recombinant pgx2 gene product, a PGase having protopectinase activity. This enzyme was a monomeric protein of 41 kDa, with an isoelectric point of pH 6.1. The characteristics of this PGase substantially coincide, with that of recombinant pgx2 gene product, and the PGase is assumed to be native pgx2 gene product. The production of PGase-X2 was confirmed to be regulated by ambient pHs.     

Biological and biochemical differences between variants of spleen focus-forming virus can be localized to a region containing the 3'' end of the envelope gene.

Two variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, induce acute erythroleukemia in mice but differ in their effects on erythroid cells as well as in the posttranslational modification of the product of their envelope genes. To localize the region of the SFFV envelope gene responsible for these differences, we utilized a recombinant virus containing the 3' half of the SFFVP env gene, where the vast majority of the differences between SFFVP and SFFVA reside, and the SFFVP long terminal repeat (LTR) on an SFFVA background. Analysis of the recombinant virus indicates that it is capable of inducing all of the biological effects previously associated with SFFVP, including the ability to proliferate and differentiate without the need for erythropoietin. In addition, the env gene product of the recombinant virus can be detected on the cell surface, a property previously associated only with SFFVP. Although the recombinant virus also contains LTR sequences from SFFVP, we do not believe it is likely that the four LTR nucleotides that are unique to SFFVP are responsible for the biological or biochemical differences observed. These results strengthen the argument that the SFFVP env gene product acts at the cell surface to alter the hormonal requirements for erythroid cell growth and differentiation.     

Industrial production of heterologous proteins by fed-batch cultures of the yeast Saccharomyces cereuisiae

Abstract: This review concerns the issues involved in the industrial development of fed-batch culture processes with Saccharomyces cereriviae strains producing heterologous proteins. Most of process development considerations with fed-batch recombinant cultures are linked to the reliability and reproducibility of the process for manufacturing environments where quality assurance and quality control aspects are paramount. In this respect, the quality, safety and efficacy of complex biologically active molecules produced by recombinant techniques are strongly influenced by the genetic background of the host strain, genetic stability of the transformed strain and production process factors. An overview of the recent literature of these culture-related factors is coupled with our experience in yeast fed-batch process development for producing various therapeutic grade proteins. The discussion is based around three principal topics: genetics, microbial physiology and fed-batch process design. It includes the fundamental aspects of yeast strain physiology, the nature of the recombinant product, quality control aspects of the biological product, features of yeast expression vectors, expression and localization of recombinant products in transformed cells and fed-batch process considerations for the industrial production of Saccharomyces cerevisiae recombinant proteins. It is our purpose that this review will provide a comprehensive understanding of the fed-batch recombinant production processes and challenges commonly encountered during process development.     

牛分枝杆菌MPB83基因的原核表达及免疫活性分析

利用PCR技术,以牛型分枝杆菌(M.bovis)Vallee菌株的全基因组DNA为模板,扩增出了一条600bp的MPB83基因片段,将其克隆至pMD18T载体中,经核苷酸序列测定确证后,KpnI/EcoRI双酶切,然后亚克隆到原核表达载体pET30a的相同酶切位点,构建表达质粒pETMPB83,将鉴定的重组质粒转化大肠杆菌BL21(DE3),IPTG诱导后SDSPAGE检测表达情况,重组质粒pETMPB83在30kDa处有一特异表达带,与预计大小相符。经Ni柱纯化后,Westernblot检测纯化蛋白具有免疫活性,用纯化的该蛋白进行动物(兔)接种制备抗血清,用Westernblot和ELISA检测该抗血清的效价和特异性,结果表明特异性较好。