Isolation of recombinant proteins from culture broth by co-precipitation with an amino acid carrier to form stable dry powders.
Protein-coated microcrystals can be generated by co-precipitation of protein and a water-soluble crystalline carrier by addition to excess water miscible organic solvent. We have investigated this novel process for its utility in the concentration and partial purification of a recombinant protein exported into the culture broth during expression by Pichia pastoris. Co-precipitation with a L-glutamine carrier selectively isolated the protein content of the culture broth, with a minimal number of steps, and simultaneously removed contaminants including a novel yeast metabolite. This pigment co-elutes during aqueous chromatography but its elucidation as a benzoylated glycosamine suggested a simple route of removal by partition during the co-precipitation process. Scale-up of the process was readily achieved through in-line mixing and subsequent reconstitution of the dried protein-coated microcrystals yielded natively folded, bioactive protein. Additional washing of the crystals with saturated L-glutamine facilitated further purification of the recombinant protein immobilized on the L-glutamine carrier. Thus, we present a novel method for the harvesting of recombinant protein from culture broth as a dry powder, which may be of general applicability to bioprocessing.
Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK. b.d.moore@strath.ac.uk
Barry D
BD
Moore
Joseph
J
Deere
RuAngelie
R
Edrada-Ebel
Andrew
A
Ingram
Christopher F
CF
van der Walle
D011232:Chemical Precipitation; D003470:Culture Media / Q000737:chemistry*; D003890:Desiccation; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism*; D011208:Powders; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D003470:Culture Media; D005656:Fungal Proteins; D011208:Powders; D011994:Recombinant Proteins; D005973:Glutamine