Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein.
The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tended to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutics is currently under exploration.
Zhang
Lin
L
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Narayanan
Niju
N
Brand
Stephen R
SR
Nicolette
Charles A
CA
Baroja
Miren
M
Arp
Jacqueline
J
Wang
Hao
H
Moo-Young
Murray
M
Chou
C Perry
CP
D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D014644:Genetic Variation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000627:therapeutic use; D012694:Serine / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism; D000097802:CD83 Antigen
D015703:Antigens, CD; D004220:Disulfides; D005982:Glutathione Transferase; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D012694:Serine; D014841:von Willebrand Factor; D000097802:CD83 Antigen