| dc.description.abstract | For bacteria, the most common expression system for recombinant protein production is Escherichia coli (E. coli). However, a large fraction of recombinant proteins produced in E. coli are in an insoluble form which sometimes makes them irrelevant for therapeutic applications. There could be several advantages in producing recombinant proteins in other bacteria, such as Corynebacterium glutamicum (C. glutamicum), especially when it comes to proteins that are difficult to secrete or dependent on effective downstream processes. C. glutamicum has an ability to secrete proteins into the growth medium, and has a different intercellular environment which could result in soluble protein expression when E. coli fails. The aim of this thesis was, therefore, to test and adapt the expression technology of Vectron Biosolutions, the XylS/Pm expression cassette, to the production of recombinant proteins in C. glutamicum.
Two C. glutamicum/E. coli shuttle vectors were constructed. Both of them, pXMJ19- mCherry and pVB-4A0E1-mCherry harboring the XylS/Pm expression cassette, were found to be functional in E. coli, producing mCherry in high amounts when induced with IPTG and m-toluate respectively, yielding pink cultures. For both of them, most of the mCherry protein was found in a soluble state.
Both vectors were successfully transferred into C. glutamicum. However, none of them resulted in mCherry production. To further investigate why no mCherry protein was ob- tained, and to possibly figure out if the bottleneck was at xylS or mCherry level and also if transcription or translation of these proteins were the main issue, the transcript lev- els of mCherry and xylS were evaluted. For C. glutamicum harboring pXMJ19-mCherry, mCherry transcriptis could be identified and transcript level increased 12 times when the culture was induced with IPTG. However, the amount of mCherry transcript was signifi- cantly less than for E. coli. For C. glutamicum harboring pVB-4A0E1-Cherry, no mCherry transcript could be identified. From this, it could not be confirmed whether mCherry is a suitable reporter gene in C. glutamicum. The results also showed that when compared to E. coli, the amount of xylS transcript from the XylS/Pm expression cassette in C. glutamicum was very low.
Even though no functional shuttle vector expressing recombinant proteins from the XylS/Pm expression cassette has been verified, xylS transcription has been identified as a bottleneck for protein expression from the XylS/Pm expression cassette in C. glutamicum. This work is a contribution to the ongoing research into developing C. glutamicum as an alternative bacterial host. | |
