Evaluation of pretreatment methods for primary recovery and capture of an antibody fragment (alpha CD22scFv) from Chlamydomonas reinhardtii lysates Academic Article uri icon


  • 2015. Optimum conditions for extraction of an acidic (pI 5.1) single-chain antibody fragment (CD22scFv) produced in the chloroplast of Chlamydomonas reinhardtii were determined as function of pH, NaCl, and Tween 20. The maximum extraction yield of CD22scFv 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 CD22scFv. 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 CD22scFv recovery yield compared to the other two methods. To check compatibility of pretreated extracts with non-affinity adsorption chromatography, batch adsorption experiments of CD22scFv from pretreated extracts were performed. Ammonium sulfate (1 M) pretreated extracts were not suitable for direct capture of CD22scFv by phenyl sepharose resin because the target protein could not be adsorbed completely even in the presence of 1.5 M 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 chromatography of CD22scFv from chitosan- and acid-pretreated extracts resulted in 20- and 13-purification fold, respectively.

published proceedings


author list (cited authors)

  • Munjal, N., Kulkarni, S., Quinones, K., Tran, M., Mayfield, S. P., & Nikolov, Z. L.

citation count

  • 2

complete list of authors

  • Munjal, Neera||Kulkarni, Sayali||Quinones, Katelyn||Tran, Miller||Mayfield, Steve P||Nikolov, Zivko L

publication date

  • January 2015