Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein.
An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (1) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10% of total cellular protein, with a cell density of 8 OD(600) was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed.
Xu
Yali
Y
Department of Chemical Engineering, University of Waterloo, 200 University Ave. West, Waterloo, Ont. N2L3G1, Canada.
Zhang
Lin
L
Yao
Weifang
W
Yedahalli
Shreyas S
SS
Brand
Stephen
S
Moo-Young
Murray
M
Perry Chou
C
C
D015703:Antigens, CD / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use; D006801:Humans; D007136:Immunoglobulins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000627:therapeutic use; D000097802:CD83 Antigen
D015703:Antigens, CD; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D000097802:CD83 Antigen