Harris, Robert Peter; (2002) Proteomic approaches for optimal production and isolation of recombinant proteins. Doctoral thesis (Ph.D), UCL (University College University).

Text Proteomic_approaches_for_optim.pdf Download (19MB)

Abstract

It is important to perform fermentations to produce recombinant proteins under conditions where the host proteins which are potential contaminants are at the lowest possible level without compromising expression of the product. One of the ways this can be achieved is to adjust the temperature and media. Proteomic approaches using 2-D electrophoresis represent a rapid way in which the properties relevant to separation of the product protein and the potential host protein contaminants can be assessed. This study investigates the application of proteomic technology in the production and isolation of recombinant proteins expressed within E.coli. In the first instance the effect of operating temperature on the production of hepatitis C virus (HCV) proteinase was investigated. It was found that the proteome of E.coli was not static, but responsive to the bioreactor environment. The effect of operating temperature was also investigated on the production of glutathione S-transferase (GST). Again, the proteome was shown to be responsive, and host protein contaminants exhibiting similar characteristics of pi and Mw to GST were found to be lowest at 30C. Isolation of GST from these fermentations confirmed that host protein contamination was lower with the 30C fermentation, without compromising the expression of the product. Additional studies of the effect of fermentation media selection showed that the proteome of a native strain of E.coli (DH5 ) changed significantly with different media. Cell culture with a glycerol-rich media was found to produce the least host protein contamination of proteins with similar characteristics to GST, which was confirmed by isolation of specific proteins. Using this proteomic analysis, the conditions for optimal GST production were defined. The effect of recombinant protein expression on the E.coli proteome was also investigated. It was found that recombinant protein production within the host cell affected the proteome make-up, and changed levels of numerous proteins. Proteomics represents a powerful insight into the recombinant host metabolome when expressing a target protein. This thesis is an illustration of the potential to exploit this, in order to produce rapid ways in which protein production can be made more optimal. It also includes a novel way of representing proteomic data for this purpose.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item