共查询到20条相似文献,搜索用时 0 毫秒
1.
Mass production of human epidermal growth factor using fed-batch cultures of recombinant Escherichia coli 总被引:2,自引:0,他引:2
Shimizu N Fukuzono S Harada Y Fujimori K Gotoh K Yamazaki Y 《Biotechnology and bioengineering》1991,38(1):37-42
Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively. 相似文献
2.
Ryoichi Tanaka Masahiro Hashimoto Matsujiro Ishibashi Hiroko Tokunaga Satoru Taura Makoto Mizukami Akira Miyauchi Masao Tokunaga 《Biotechnology letters》2001,23(22):1853-1857
DsbA and DsbC, members of the thioredoxin super-family of redox proteins, which are expressed in the periplasmic space of Escherichia coli, were cloned into and successfully secreted from Brevibacillus choshinensis at 100 g ml–1. Both proteins were active in exchanging disulfide bonds of bovine insulin in vitro. Furthermore, DsbA secreted by B. choshinensis promoted the conversion of non-native human epidermal growth factor to the native form. 相似文献
3.
Sharma K Babu PV Sasidhar P Srinivas VK Mohan VK Krishna E 《Protein expression and purification》2008,60(1):7-14
Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 × 105 IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results. 相似文献
4.
AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins. 相似文献
5.
L. B. Tsai M. Mann F. Morris C. Rotgers D. Fenton 《Journal of industrial microbiology & biotechnology》1987,2(3):181-186
Summary Two kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period. 相似文献
6.
7.
Zhi-Yong Zheng Shan-Jing Yao Xiaobei Zhan Chi Chung Lin 《Biotechnology and Bioprocess Engineering》2009,14(1):52-59
Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant
Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate
was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved.
With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation
of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell
respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition
of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch
cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently,
the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect
of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined.
A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high
level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls. 相似文献
8.
B H Chung Y J Choi S H Yoon S Y Lee Y I Lee 《Journal of industrial microbiology & biotechnology》2000,24(2):94-99
Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated
in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the
mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production.
Under this condition, 2.8 g L−1 of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification
of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein
expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography,
refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities
of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99.
Received 13 January 1999/ Accepted in revised form 02 October 1999 相似文献
9.
Recombinant Escherichia coli strain C600/pBV-TRAIL (encoding for 114-281 amino acids of TRAIL's soluble fragment) produced a recombinant human tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Using a combined strategy of exponential feeding and pH-stat feeding, high concentrations of biomass (65 g dry wt l(-1)) and active soluble TRAIL (1.4 g l(-1)) were obtained within 30 h. The accumulation of acetate, which usually occurs during the process of high-density culture of Escherichia coli and especially in the induction stage of protein synthesis, was avoided. 相似文献
10.
11.
Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli 总被引:5,自引:0,他引:5
Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively. 相似文献
12.
M. E. Gasparian P. A. Elistratov N. I. Drize I. N. Nifontova D. A. Dolgikh M. P. Kirpichnikov 《Biochemistry. Biokhimii?a》2009,74(2):221-225
Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity. 相似文献
13.
William F. Greenlee Karen M. Dold Rosemarie Osborne 《In vitro cellular & developmental biology. Plant》1985,21(9):509-512
Summary In humans, the skin is a particularly sensitive target for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and certain halogenated analogs. Reported lesions include a thickening of the epidermis (acanthosis), hyperkeratosis,
and squamous metaplasia of the epithelial lining of the sebaceous glands. In this report we describe ongoing studies on the
actions of TCDD on cultured human epidermal cells. This system has been established as an in vitro model for interfollicular
epidermal hyperkeratinization. Treatment of newly confluent cultures with TCDD results in enhanced differentiation as judged
by histologic examination of the cultures, a decrease in the number of basal proliferating cells, and an increase in the number
of envelope competent (differentiating) cells and terminally differentiated cells with highly cross-linked cornified envelopes.
Changes in the differentiation program are preceded by a decrease in epidermal growth factor (EGF) binding. The concentration
dependence and stereospecificity for these responses suggest the involvement of theAh receptor. We propose that TCDD modulates normal patterns of epidermal differentiation through direct actions on proliferating
basal cells, modulating the responsiveness of these cells to growth factors such as EGF.
This paper was presented at the Session-In-Depth on In Vitro Applications in Toxicology at the 34th Annual Meeting of the
Tissue Culture Association, Orlando, FL, June 12–16, 1983. Rosemarie Osborne was a Chemical Industry Institute of Toxicology
Postdoctoral Fellow. 相似文献
14.
15.
Kim Il-Chul Kim Chang-Ho Hong Suk-In Kim Seung-Wook 《World journal of microbiology & biotechnology》2001,17(9):869-872
In order to obtain high productivity of clavulanic acid, a newly-introduced carrier, polyurethane pellet (PUP) Z97-020 was used for the immobilization process. In a stirred-tank bioreactor, batch cultivation by Streptomyces clavuligerus KK immobilized on PUP Z97-020 gave about 3100 mg of clavulanic acid per litre, representing an increase of 200% in productivity compared with that by fed-batch cultivation of free cells (1500 mg/l). However, the clavulanic acid produced rapidly decomposed due to the pH change during batch cultivation. Fed-batch cultivation by immobilized S. clavuligerus KK gave an excellent level of clavulanic acid up to 3250 mg/l, a productivity increase of 220% compared with that by fed-batch cultivation of free cells. These results suggest that immobilization with PUP Z97-020 is a more effective process for the production of clavulanic acid and that the maintenance of pH by fed-batch cultivation with glycerol as a limiting substrate prevents the clavulanic acid from decomposing during the fermentation. 相似文献
16.
Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role
in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential
reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated
dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production,
we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58
g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation,
subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout
the fermentation. 相似文献
17.
Effects of iron-limitation ofEscherichia coli on growth,the respiratory chains and gallium uptake 总被引:1,自引:0,他引:1
Julia A. M. Hubbard Krystyna B. Lewandowska Martin N. Hughes Robert K. Poole 《Archives of microbiology》1986,146(1):80-86
The effects of iron limitation on growth, the composition and function of the respiratory chains, and gallium uptake inEscherichia coli have been studied. Decreasing the iron concentration in a defined medium using Chelex resin gave lower growth yields in both continuous culture and prolonged batch culture. In the former, ironlimited (entering [Fe]2.0 M) cells exhibited diminished respiration rates, respiration-driven proton translocation quotients, and levels of non-haem iron and cytochromes. The cellular concentration of haemoproteinb-590 (a cytochromea
1-like hydroperoxidase) decreased 20-fold on iron limitation, whilst a CO-binding pigment with an absorption maximum in the dithionite-treated form near 500 nm appeared. Gallium(III) (9 M) added to iron-limited, but not iron-sufficient, cultures diminished growth yields further; cells grown with low entering concentrations of iron took up less gallium than iron-sufficient cells. These results are attributed to the interference by gallium(III) with siderophore-mediated metal uptake. Gallium also stimulated iron uptake and was itself accumulated by iron-sufficient cells, suggesting that gallium(III) also affects the iron transport system(s) of low affinity. 相似文献
18.
Bu-Youn Kim Jong-Chul Kim Hyune-Hwan Lee Hyung-Hwan Hyun 《Biotechnology and Bioprocess Engineering》2001,6(1):11-17
To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited
level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both
increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold
compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when
using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture. 相似文献
19.
Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes). A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)2 and trimer (DHBS)3, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid. The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids. Enterobactin was found to be the predominant product in all mutant strains. The mutant strains behaved differently with regard to the breakdown products. All degradation products, such as DHBS, (DHBS)2 and (DHBS)3, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant. From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not. Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e. DHBS, (DHBS)2 and (DHBS)3, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer. Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not. 相似文献
20.
Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin 总被引:2,自引:0,他引:2
Janice E. Errick Katherine W. A. Ing Margaret C. Eggo Gerard N. Burrow 《In vitro cellular & developmental biology. Plant》1986,22(1):28-36
Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and
to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture
conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of
insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine
(10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present
at 10−9
M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system
provides a means to study the hormonal control of growth and differentiation in human thyroid cells.
This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto;
and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow. 相似文献