Molecular manipulation associated with disulfide bond formation to enhance the stability of recombinant therapeutic protein.
Cys²⁷ in the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein, was identified as a target for molecular manipulation. Two Escherichia coli strains of BL21(DE3) and Origami B(DE3), respectively, with a reducing and an oxidative cytoplasm were used as the expression host to produce the Cys²⁷ mutants. It was observed that Cys²⁷ was involved in the in vivo formation of intramolecular disulfide bonds when hCD83ext was expressed in Origami B(DE3). The Origami-derived protein products had a higher tendency than the BL21-derived counterparts for multimerization via the in vitro formation of intermolecular disulfide bonds. Various analyses were conducted to identify the structural differences among these mutant variants. Most importantly, molecular stability was enhanced by the Cys²⁷ mutations since the Cys²⁷ mutants derived from either BL21 or Origami were much less susceptible to degradation compared to wild-type hCD83ext. This study highlights the implications of aberrant disulfide bond formation on the production of therapeutic proteins.
Zhang
Lin
L
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1.
Moo-Young
Murray
M
Chou
C Perry
CP
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000097802:CD83 Antigen
D015703:Antigens, CD; D003545:Cysteine; D004220:Disulfides; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D000097802:CD83 Antigen