首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
In spite of the generally stable nature of immobilized perfusion culture, its profile of target protein production frequently shows variations. This might be explained by the drift in the metabolism of cultured cells. To address this issue, we performed a set of four Opticell bioreactor cultures producing recombinant anticogulant protein PCGFX. All the cultures lasted 40-50 days with the oxygen consumption rate (OCR) mostly around 10 μmol min−1; nevertheless, glucose and lactate metabolism was fluctuated with a parallel fluctuation in the recombinant protein productivity (RPP). The mean productivity of recombinant PCGFX was determined to be about 1.0 mg day−1 for all the cultures. The statistical analysis revealed a significant correlation between the lactate production rate (LPR) and RPP in two cultures. A significant correlation was further found between average OCR and RPP in another culture where OCR was exceptionally lowered under serum-free conditions. No parameter significantly correlated with RPP in the remaining one culture; thus, the overt drift of RPP resulted, at least partly, from that of the cell metabolic activity and the present data should be helpful to explore a strategy for maximizing productivity.  相似文献   

3.
Methods to increase the production of recombinant proteins in mammalian cell cultures have been developed which reduce in-culture growth through prohibiting progression of the cell cycle. This arrest increases the proportion of cells in the G1-phase of the cell cycle, and subsequently increases their specific productivity (QP). Through careful balancing of the decreased growth rate with an increased QP, multi-fold increases in recombinant protein yield can be achieved.  相似文献   

4.
Baculovirus vector systems are extensively used for the expression of foreign gene products in insect and mammalian cells. New advances increase the possibilities and applications of the baculovirus expression system, which makes it possible to express multiple genes simultaneously within a single infected insect cell and to obtain multimeric proteins functionally similar to their natural analogs. Recombinant viruses with expression cassettes active in mammalian cells are used to deliver and express genes in mammalian cells in vitro and in vivo. Further improvement of the baculovirus expression system and its adaptation to specific target cells can open up a wide variety of applications. The review considers recent achievements in the use of modified baculoviruses to express recombinant proteins in eukaryotic cells, advantages and drawbacks of the baculovirus expression system, and ways to optimize the expression of recombinant proteins in both insect and mammalian cell lines.  相似文献   

5.
The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.  相似文献   

6.
The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.  相似文献   

7.
Actinomycetes (Actinobacteria) are highly attractive as cell factories or bioreactors for applications in industrial, agricultural, environmental, and pharmaceutical fields. Genome sequencing of several species of actinomycetes has paved the way for biochemical and structural analysis of important proteins and the production of such proteins as recombinants on a commercial scale. In this regard, there is a need for improved expression vectors that will be applicable to actinomycetes. Recent advancements in gene expression systems, knowledge regarding the intracellular environment, and identification and characterization of plasmids has made it possible to develop practicable recombinant expression systems in actinomycetes as described in this review.  相似文献   

8.
Hirudin, a blood anticoagulant protein from leeches, and β-glucuronidase were produced in Brassica carinata Braun (Ethiopian mustard) seeds using oleosin as a carrier. Cotyledonary petioles were infected with Agrobacterium strains containing oleosin-glucuronidase (pCGNOBPGUS-A) or oleosin-hirudin (pCGN-OBHIRT) constructs. Polymerase chain reaction and neomycin phosphotransferase II enzyme assays confirmed the presence of the fusion genes in plants regenerating under selection. The fusion polypeptides were correctly expressed and targeted to the oil-bodies of the seeds with high fidelity (ca. 90%). Recombinant protein was purified from all other cellular protein by a simple flotation process and cleaved from oil-bodies using the endoprotease, Factor Xa. Hirudin activity was measured using a colorimetric thrombin inhibition assay and an activity in the range of 0.2–0.4 antithrombin units per milligram of oil-body protein was detected. B. carinata offers an attractive alternative for the production of recombinant proteins using oleosin technology. Received: 20 March 1997 / Revision received: 5 June 1997 / Accepted: 30 July 1997  相似文献   

9.
10.
Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.  相似文献   

11.
Recombinant proteins (r-proteins) are increasingly important in fundamental research and for clinical applications. As many of these r-proteins are of human or animal origin, cultivated mammalian cells are the host of choice to ensure their functional folding and proper posttranslational modifications. Large-scale transfection of human embryonic kidney 293 or Chinese hamster ovary cells is now an established technology that can be used in the production of hundreds of milligram to gram quantities of a r-protein in less than 1 mo from cloning of its cDNA. This chapter aims to provide an overview of large-scale transfection technology with a particular emphasis on calcium phosphate and polyethylenimine-mediated gene transfer.  相似文献   

12.
We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell Une, 9–186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell Unes, which resulted in a 4–5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration  相似文献   

13.
High level expression of a recombinant gene results in growth arrest, followed by overgrowth by non-productive derivatives. Two methods are described for the isolation of E. coli BL21* strains that are improved hosts for recombinant protein production. Both are based upon the observations (i) that fluorescence of a C-terminal GFP tag is a reliable reporter of the production and correct folding of the N-terminal target domain; and (ii) rare mutants arise spontaneously that remain productive during long periods of high level recombinant protein production. The first method relies upon identifying these mutants amongst colonies on agar plates; the other exploits fluorescence activated cell sorting. Although identical mutations in the regulatory region of the T7 polymerase gene were found in all of the improved host strains isolated, they differed in their ability to accumulate the outer membrane protein, Ccp, or a cytoplasmic protein, CheY-GFP. Cytochrome c peroxidase activity of recombinant Ccp from one of these strains was demonstrated. Changes in levels of T7 polymerase expression are therefore insufficient to ensure increased accumulation of all recombinant proteins. We demonstrate that the methods described allow strains to be isolated that carry other, currently uncharacterised mutations that are required depending on the target protein.  相似文献   

14.
Production of recombinant proteins by yeast cells   总被引:2,自引:0,他引:2  
Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.  相似文献   

15.
Jäger V 《Cytotechnology》1996,20(1-3):191-198
Conclusion High density perfusion culture of insect cells for the production of recombinant proteins has proved to be an attractive alternative to batch and fed-batch processes. A comparison of the different production processes is summarized in Table 3. Internal membrane perfusion has a limited scale-up potential but appears to the method of choice in smaller lab-scale production systems. External membrane perfusion results in increased shear stress generated by pumping of cells and passing through microfiltration modules at high velocity. However, using optimized perfusion strategies this shear stress can be minimized such that it is tolerated by the cells. In these cases, perfusion culture has proven to be superior to batch production with respect to product yields and cell specific productivity. Although insect cells could be successfully cultivated by immobilization and perfusion in stationary bed bioreactors, this method has not yet been used in continuous processes. In fluidized bed bioreactors with continuous medium exchange cells showed reduced growth and protein production rates.For the cultivation of insect cells in batch and fedbatch processes numerous efforts have been made to optimize the culture medium in order to allow growth and production at higher cell densities. These improved media could be used in combination with a perfusion process, thus allowing substantially increased cell densities without raising the medium exchange rate. However, sufficient oxygen supply has to be guaranteed during fermentation in order to ensure optimal productivity.  相似文献   

16.
Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15–22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA. © 1993 Wiley-Liss, Inc.  相似文献   

17.
A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.  相似文献   

18.
A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.  相似文献   

19.
Repair of DNA alkylation adducts in mammalian cells   总被引:2,自引:0,他引:2  
Carcinogenic alkylating agents, including nitrosamines, are able to alkylate DNA at various sites. This review presents evidence of the high degree of specificity in the type of DNA damage induced by various N-nitroso compounds and in the DNA repair processes among tissues or cells of different species. The O6-alkylguanine DNA alkyltransferase activity in various human and rodent tissues is discussed as well as the detection of O6-methylguanine in human DNA, using monoclonal antibodies and radioimmunoassay. The relevance of these findings to the mechanisms of cancer induction by nitrosamines is discussed.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号