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Please use this identifier to cite or link to this item: http://eprint.iitd.ac.in/handle/2074/421

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dc.contributor.authorGupta, Pankaj-
dc.contributor.authorSahai, Vikram-
dc.contributor.authorBhatnagar, Rakesh-
dc.identifier.citationBiochemical and biophysical research communications, 285, 1025-1033en
dc.description.abstractHigh cell density cultivation has been one of the most effective ways to increase cell as well as the product yields. The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as fusion protein with 63 histidine residues under the transcriptional regulation of the T5 promoter in Escherichia coli. Various strategies were tried to scale up the expression of the recombinant lethal factor by bioprocess optimization using fed batch culture technique in a 14 litre fermentor. The media, a defined mixture of salts, trace elements, vitamins,etc. along with a specified carbon source was used for the growth. The pH of the media was maintained at 6.8 while the temperature was changed from 37 to 28°C during the cultivation. During the growth and induction phases, the DO was maintained above 20% by automatic control of agitation. The specific growth rate was controlled by utilizing an exponential feeding profile determined from mass balance equations.As a result of control of specific growth rate at two different levels, there was about twenty five fold increase in biomass compared to the biomass in the shake flask. E. coli cells yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa.The protein was purified to homogeneity using metal chelate affinity chromatography, followed by anion exchange on FPLC using Mono-Q column. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinity. The recombinant LF resembled the LF purified from B. anthracis in the macrophage lysis assay, using a murine macrophage cell line J774A.1 sensitive to anthrax toxin. It was possible to achieve a yield of 50 mg of the purified protein from 1 litre culture broth.en
dc.format.extent2396253 bytes-
dc.subjectdensity cultivationen
dc.subjectshake flasken
dc.subjectcytosolic proteinen
dc.subjectMono-Q columnen
dc.subjectmurine macrophageen
dc.titleEnhanced expression of the recombinant lethal factor of bacillus anthracis by fed-batch cultureen
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