The eukaryotic green alga, Chlamydomonas reinhardtii, is a unique expression platform that can efficiently express complex therapeutic proteins. However, demonstrating that therapeutic molecules can be produced in quantifiable levels is essential to establish the potential of the C. reinhardtii expression system. Thus, the objective of this investigation was to determine the process conditions that could maximize C. reinhardtii biomass accumulation and induced-production of the two recombinant proteins, a single chain fragment antibody molecule (αCD22 scFv) and malaria vaccine antigen (Pfs25), produced in the chloroplast of C. reinhardtii. To achieve a higher production of recombinant proteins, cultivation variables of C. reinhardtii, such as mixing, light-induction time and intensity, nutrient depletion and culture age, were investigated and optimized. The optimal light-induction time was 24 h at a light intensity of 300 μmol m −2 s −1. Replacement of the culture media in the late exponential growth with fresh media was beneficial to the accumulation of recombinant proteins. Optimization led to increases in the accumulation of recombinant proteins by six-fold and the recombinant protein fraction in the extracted soluble protein by twofold .

Amygdalin-degrading lactobacilli with practical application in the biodetoxification of amygdalin were characterized. These strains show potential for use as starter cultures to improve the safety of foods and feeds.

Optimum conditions for extraction of an acidic (pI 5.1) single-chain antibody fragment (αCD22scFv) produced in the chloroplast of C. reinhardtii were determined as function of pH, NaCl, and Tween 20. The maximum extraction yield of αCD22scFv was achieved at pH 8 with 400 mM salt and 0.5% Tween. The effectiveness of mechanistically different precipitation methods (polymer, isoelectric, and salt) for reduction of DNA, chlorophyll, and host cell protein in cell lysates and clarified cell lysates was compared. Acid and ammonium sulfate precipitation significantly reduced host cell protein and chlorophyll in cell lysates and clarified cell lysates with a minimal loss of αCD22scFv. Precipitation of cell lysates and clarified cell lysates with chitosan (cationic polymer) was the best overall pretreatment method and was accompanied by 10-15% lower αCD22scFv recovery yield compared to the other two methods. To check compatibility of pretreated extracts with non-affinity adsorption chromatography, batch adsorption experiments of αCD22scFv from pretreated extracts were performed. Ammonium sulfate (1M) pretreated extracts were not suitable for direct capture of αCD22scFv by Phenyl Sepharose resin because the target protein could not be adsorbed completely even in the presence of 1.5M ammonium sulfate. CD22scFv completely adsorbed onto Capto Q resin at pH 8 from acid-and chitosan-pretreated extract after reducing the extracts ionic strength to 4 mS. Capture