For the production of recombinant proteins, the major challenge lays in mMaximising the yield of biologically functional proteins is still a challenge for recombinant protein production. In the most commonly used host Escherichia coli, proper protein folding is compromised by the formation of inclusion bodies, i.e. aggregated deposits of denatured proteins. In this thesis, the production of active α-glucosidase from the yeast Saccharomyces cerevisiae was optimised in two aspects. First the cultivation parameters that optimise the productivity were determined and their influence on the cultivation process was analysed. Second the new approach of reactivating the inclusion bodies inside the living cell was studied. Cultivations processes implementing this approach were developed in bench-top and pilot scale for production of α-glucosidase and the influence of cultivation parameters on the productivity was analysed. Inclusively, Specifically the main part of the thesis was concentrated on investigating the roles of the small heat shock proteins of E. coli, IbpA and IbpB, in the metabolism of inclusion bodies and in cell protection metabolismwere investigated. Small heat shock proteins IbpA and IbpB have been found tightly associated with inclusion bodies of numerous recombinant proteins and were suggested implicated to promote the rapidchange the kinetics of inclusion bodies disaggregation. α-Glucosidase from the yeast Saccharomyces cerevisiae was chosen as model protein throughout the work due to some reasons, first the yield of soluble α-glucosidase and the formation of inclusion bodies is strongly depended on cultivation parameters. Second the ibpAB operon is induced during recombinant α-glucosidase production, which indicates the enhanced need for IbpA and IbpB. Last the native state of α-glucosidase can be easily monitored by enzymatic assay. Important parameters that influence the α-glucosidase folding are temperature and growth rate. Temperatures that maximise the growth rate almost extensively promote aggregation of α-glucosidase, while temperatures of 30°C or below were compatible with production of active α-glucosidase. Moreover, as the growth rate is a function of temperature in shake flask experiments, maximum specific α-glucosidase activities were obtained at 24°C. But since the growth was decelerated at lower temperature, the volumetric activities resulting from specific activity and cell density were equally high at 24°C and 30°C. The influence of cultivation parameters was studied in shake flasks and in bioreactors. The fFed-batch cultivations with exponentially increasing feeding rates to enable culture growth with the constant growth rates. of the cultures could isolate the effect of specific growth rate of that of temperature. The inverse correlation of inclusion bodies formation with specific growth rate was observed at the set specific growth rate µset in the range from 0.06 h-1 to 0.22 h-1, while the yield of active product was inversely correlated only in the range of specific growth rate higher than 0.12 h-1. The specific growth rate set by the feeding rate had a strong impact on the accumulation of active α-glucosidase. The specific activity was six fold higher at low growth rate (µset 0.12 h-1) than at higher growth rate (µset > 0.18 h-1) at 37°C. As slow growth affects biomass formation, the highest volumetric product yield and productivity were achieved with µset = 0.12 h-1. Disaggregation of inclusion bodies and accumulation of active α-glucosidase could be achieved by arrest of protein synthesis and/or reduction of the cultivation temperature. In shake flasks. The new protocol for production by in vivo reactivation from inclusion bodies by temperature downshift was studied, based on the possibility of inclusion bodies to disintegrate. Tthe reactivation efficiency depended on the temperature during aggregation the production and reactivation phases anddisaggregation, was more efficient highest with production at aggregation temperature of 42°C followed by reactivation and disaggregation temperature of at 30°C. By this optimised protocol, tThe final activitye yield five hours after production by this new protocol under optimised conditions was improved in shake flask and on complex LB medium was improved twofold relative to highest value by direct production at 24°Cwithout temperature downshift despite at temperature of 30°C, which was optimum for productivity of active α-glucosidase. In fed-batch cultivations on defined medium, the α-glucosidase production was very efficient: while the specific activity after five hours production reached in shake flasks experiments on complex medium 3 U mg-1 in direct production without reactivation and rose to 4.5 U mg-1 with reactivation under optimised temperature profiles, it reached already 6 U mg-1 in fed-batch cultivation under optimised condition after the same time of production and rose to 9 U mg-1 on the end of cultivation (18 h production). Since the optimised conditions for in vivo reactivation in fed-batch cultivations were the same as for direct production, the highest yield of α-glucosidase activity was achieved by temperature reduction at the same time of induction. The role of IbpA and IbpB in the metabolism of inclusion bodies, formed during α-glucosidase production was studied. While ibpAB has been found dispensable up to temperatures of 50°C, growth during α-glucosidase production was impaired in the ibpAB deletion strain. Also tThe heat shock response, which is induced during α-glucosidase production, was intensified, most obviously with the disaggregation chaperones ClpB and DnaK, and correspondingly was suppressed attenuated by overexpression of ibpAB, whereas in the ibpAB deletion strain, concomitant with growth impairment, the heat shock response was intensified, most obviously with the disaggregation chaperones ClpB and DnaK. The small heat-shock proteins thus exert an important function in cell protection under conditions of strong protein aggregation. The role of IbpA and IbpB in the metabolism of inclusion bodies formed during α-glucosidase production was also studied. After arrest of protein synthesis, α-glucosidase inclusion bodies were dissaggregated, reactivated and degraded in a clpB and dnaK dependent process. The disaggregation and degradation was decelerated by small heat shock protein IbpA/IbpB in a temperature-dependent manner. The With a high production temperature of 42°C, overexpression of ibpAB can prolong the reactivation process and increase the reactivation yield by 25 % compared with without ibpAB overexpression. |