Recombinant Escherichia Coli

Human insulin is derived from a biosynthetic process using strains of Escherichia coli (recombinant DNA, rDNA). Human insulin appears to cause fewer allergic reactions than does insulin obtained from animal sources. Insulin analogy, insulin lispro, and insulin aspart are newer forms of human insulin made by using recombinant DNA technology and are structurally similar to human insulin. 

Tegoni, M., and Cambillau, C., 1994. The 2.6 refined structure of the Escherichia coli recombinant Succ/turomycei cereviiiue flavocytochrome sulfite complex, Prot. Sci. 3 303n313. 

Hormones can be synthesised directly in the chemical laboratory or by inserting mammalian genes into microbes, e.g. Escherichia coli recombinant DNA technology. 

Thioacetamide induced an increase in sex-linked recessive mutations in Drosophila. It was non-mutagenic in the SalmonellalAmes mutagenicity assay, and in the Escherichia coli recombination assay. Protein synthesis in mouse hepatoma (MH-134), but not in L-929 cells, was enhanced by adding thioacetamide. 

Koyama, Y. and Furukawa, K. (1990) Qoning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Ihermus thermophilus HB27 plasmid transfer from replica-plated Escherichia coli recombinant... 

Recombinant human tumor necrosisb factor alpha from an extract of Escherichia coli Recombinant human growth hormone preparation (rhGH)... 

Amimdsen SK, Smith GR (2003) Interchangeable parts of the Escherichia coli recombination machinery. Cell 112 741-744... 

Escherichia coli, recombinant PolyOHB-co-3HV) Glucose + propionic acid 203.1 78.2 Choi and Lee (1999b)... 

Swartz JR (1996) Escherichia coli recombinant DNA technology. In Neidhardt FC (ed) Escherichia coli and Salmonella cellular and molecular biology, 2nd edn. American Society of Microbiology Press, Washington, DC,... 

Mazul, M. M. Danilov, V. S. Tetrazolium salt effect on the bioluminescence of Escherichia coli recombinant strain. Biotekhnologiya 2002, 91-96 Chem. Abstr. 2002, 138, 68140. 

Escherichia coli. The genetics of this gram-negative bacterium are very well known. For this reason, many of the first efforts to produce recombinant products from this microorganism were successful. However, because of the importance of the other criteria Hsted above, many efforts failed. E. co/i is only used to produce the milk-clotting mammalian protease chymosin [9001-98-3] (rennin). 

J. S. Pati ick and A. L. Lagu, Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC, Chem. 64 507-511 (1992). 

Another way to enhance the production of an amino acid is to make use of DNA-recombinant technology, often in combination with foe mutations already described. In this way foe negative features of foe micro-organisms are avoided. To help explain this, we will consider a well known fermentation of L-phenylalanine using Escherichia coli. We have already seen foe metabolic pathway leading to foe production of L-phenylalanine in Figure 8.4. 

The cDNA encoding the luciferase of Renilla reniformis has been obtained and expressed in Escherichia coli (Lorenz et al., 1991). The cDNA contained an open reading frame encoding a 314-amino acid sequence. The recombinant Renilla luciferase obtained had a molecular weight of 34,000, and showed an emission maximum at 480 nm in the luminescence reaction of coelenterazine, in good agreement with the data of natural Renilla luciferase. 

Miyamoto, C., Byers, D., Graham, A. F., and Meighen, E. A. (1987). Expression of bioluminescence by Escherichia coli containing recombinant Vibrio harveyi DNA. ]. Bacteriol. 169 247-253. 

Shimomura, O., and Inouye, S. (1999). The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin. Protein Expression and Purification 16 91-95. 

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Escherichia coli K12 TGI strain was used as a recipient for transformation. At studying of SOS-system activity the recombinant bioluminescent strain of Escherichia coli recA lux containing plasmid-borne fusions of the recA promoter-operator region to the Photorhabdus luminescens ZM 1 lux genes (GosNlIgenetika, Russia) was used. Increase of their luminescence in the presence of DNA damage factors [Rosen et al., 2000], were shown previously. Investigation of the luminescent response of this strain to UV radiation allows quantitatively estimate in a real time a SOS-system induction. 

Recombinant resilin production was induced, with the nonmetabolizable lactose analogue IPTG, in the Escherichia coli bacterial strain BL21(DE3)/pLysS. Cells were collected by centrifugation (10,000 g, 20 min at 4°C) and the cell pellet frozen at 80°C. 

Recently, recombinant biocatalysts obtained using Escherichia coli cells were designed for this process. The overexpression of all enzymes required for the process, namely, hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771 was achieved. These cells were used for production of a-amino acids at the concentration of above 50 g 1 dry cell weight [37]. This is an excellent example presenting the power of biocatalysis with respect to classical catalysis, since a simultaneous use of three different biocatalysts originated from one microorganism can be easily achieved. 

Recombinant DNA technology can also be used to design genes that encode for proteins with desired features [34]. The gene can be incorporated into a plasmid, which is then used to transform a bacterial host such as Escherichia coli. Finally, the production of the desired amino acid polymer is performed by the host with a precisely defined sequence and near absolute monodispersity [29, 35]. 

Comparison of whole cell biocatalytic reaction kinetics for recombinant Escherichia coli with periplasmic-secreting or cytoplasmic-expressing organophosphorus hydrolase... 

D-Aminoacid oxidase has been isolated from a nnmber of yeasts, and the nucleotide sequence of the enzyme from Rhodotorula gracilis ATCC 26217 has been established (Alonso et al. 1998). The gene could be overexpressed in Escherichia coli, and levels of the enzyme were greater under conditions of low aeration the enzyme isolated from the recombinant organisms was apparently the apoenzyme since maximum activity required the presence of FAD. 

Ratnatilleke A, JW Vrijbloed, JA Robinson (1999) Cloning and sequencing of the coenzyme B,2-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis. Reconstitution of mutase activity and characterization of the recombinant enzyme produced in Escherichia coli. J Biol Chem 274 31679-31685. 

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