Investigating the dynamics of recombinant protein secretion from a microalgal host.

Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.

New technologies for automated cell counting based on optical image analysis ;The Cellscreen'.

A prototype of a newly developed apparatus for measuring cell growth characteristics of suspension cells in micro titre plates over a period of time was examined. Fully automated non-invasive cell counts in small volume cultivation vessels, e.g. 96 well plates, were performed with the Cellscreen system by Innovatis AG, Germany. The system automatically generates microscopic images of suspension cells which had sedimented on the base of the well plate. The total cell number and cell geometry was analysed without staining or sampling using the Cedex image recognition technology. Thus, time course studies of cell growth with the identical culture became possible. Basic parameters like the measurement range, the minimum number of images which were required for statistically reliable results, as well as the influence of the measurement itself and the effect of evaporation in 96 well plates on cell proliferation were determined. A comparison with standard methods including the influence of the cultured volume per well (25 mul to 200 mul) on cell growth was performed. Furthermore, the toxic substances ammonia, lactate and butyrate were used to show that the Cellscreen system is able to detect even the slightest changes in the specific growth rate.