Špela Peternel,^1,* Vladka Gaberc-Porekar¹ and Radovan Komel¹

¹ Department of Biosynthesis and Biotransformation, National Institute of Chemistry, Ljubljana, Slovenia
* Corresponding author: E-mail: spela.peternel@ki.si;
Phon +386 1 4760262, Fax: +386 1 4760300

Abstract
As it was shown in our previous studies, significant amount of proteins trapped inside bacterial inclusion bodies (IBs) can be properly folded. Properly folded, functional proteins can be recovered from such IBs with extraction in mild, non-denaturing conditions. Such IBs were designated non-classical inclusion bodies (ncIBs). They are easy and practical resource for extraction of active proteins. In the present study, factors influencing the quality of the target protein inside IBs were studied. Green fluorescent protein (GFP) was used as the model protein, as its proper folding (activity) can be easily monitored by fluorescence. Various growth conditions for bacterial cultivation were tested in order to increase accumulation of active (fluorescent) GFP inside ncIBs.
Temperature and induction regime were recognized as very important factors affecting the growth of the bacteria Escherichia coli, as well as recombinant protein yield and protein folding. Decreasing the growth temperature resulted in higher final biomass production and slower bacterial metabolism; therefore the percentage of correctly folded target protein inside the cell was higher. In addition, the induction regime also influenced protein folding and immediate induction was found to be more suitable for production of GFP. Our studies confirm that optimization of bacterial growth conditions at all levels is very important for the production of high quality recombinant protein.

Keywords: Inclusion bodies; GFP; temperature; induction regime; fluorescence