Recombinant overexpression

The utility of MALDI-FTMS analysis for use in chemotaxonomic applications has been established, but this method can be applied to other areas of interest, such as biomedical and environmental analyses. A common method used by biochemists and biologists today is recombinant overexpression of proteins using bacterial whole cells in cases where large quantity of a protein is desired. The main method presently used to determine if the overexpression was successful is the use of SDS-PAGE (sodium dodecylsulfate-poly acrylamide gel... 

Expression of genes at a regularly low level, resulting in a low level of product, occurs in every organism. For recombinant proteins, overexpression which leads to considerably enhanced amounts of the recombinant protein is desired. Success of an attempt at recombinant overexpression cannot be predicted as of yet and depends on many factors, such as the protein to be expressed, host, vector, promoter, culture conditions, and, last but not least, the experience of the investigator. 

Recently the rat 24-OHase cDNA was recombinantly overexpressed in a bacterial cell line and reconstituted with its required cofactors [225]. Cell fractionation revealed the 24-OHase to be primarily localized in membranes, with minimum inclusion body formation, and that upon reconstitution with adrenodoxin, adrenodoxin reductase, and NADPH, 24-OHase activity was apparent. Interestingly, this preparation produced three... 

Biocataiytic Properties of Available Recombinant BVMOs The construction of recombinant overexpression systems for CHMO in E. coli and their success in regio- and enantioselective oxidation has inspired the search for other BVMOs. Several new... 

Among the more recently discovered enzymes, the NOX from Brevibacterium sp. K1309 (Bs-NOX) is noteworthy, which was purified and characterized in 2008 by Hirano and coworkers [56]. This enzyme shows a remarkable thermostability, is active from neutral to alkaline pHs, and is strongly activated by ammonium salts. However, cloning and recombinant overexpression of the gene have not been reported yet and only prehminary information about the performance of Bs-NOX... 

Pernas P, Masliah J, Olivier JL, Salvat C, Rybkine T, Bereziat G. Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release. Biochem Biophys Res Comm 19991 178 1298-1305. 

Inouye, S., et al. (1989). Overexpression and purification of the recombinant calcium-binding protein, apoaequorin./. Biochem. 105 473-477. 

Verhaegen, M., and Christopoulos, T. K. (2002). Recombinant Gaussia luciferase. Overexpression, purification, and analytical application of a bioluminescent reporter for DNA hybridization. Anal. Chem. 74 4378-4385. 

P Diketones can also be reduced diastereoselectively. Thus, a recombinant alcohol dehydrogenase from I. brevis (recLBADH) overexpressed in E. coli was used for... 

The nifU gene product (NifU) from A. vinelandii has been overexpressed in E. coli and the recombinant protein purified and characterized (60). NifU is a homodimer of 33-kDa subunits with 2 Fe atoms per subunit. Spectroscopic studies showed the presence of [Fe2S2l clusters with = 254 mV and only cysteinyl coordination, but with... 

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. 

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. 

Recombinant Whole-Cell Biocatalysts Overexpressing Catalytic Enzymes... 

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

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