Construction and expression of bi-functional proteins of single-chain Fv with effector domains.
We fused various polypeptide extensions to the C-termini of single chain Fv (scFv) and disulfide-stabilized Fv (dsFv) fragments to facilitate detection of bi-functional proteins or to add biological effector domains, which included the human metallothionein (HMT) motif and biotin mimetic sequence. These bi-functional proteins were expressed and secreted in a recombinant Pichia pastoris system and showed specific anti-idiotype binding activity, as determined by competitive radioimmunoassaying. However, the fusion protein constructed with dsFv- HMT, but not scFv-HMT, had lost this binding activity. The interruption of the structural conformation as a result in dsFv-HMT may be explained by the interactions between the cysteines engineered in dsFv domains and the cysteines in the HMT region.
Luo
D
D
Research and Development Division, Biomira Inc., Edmonton, Alberta, Canada.
Mah
N
N
Wishart
D
D
Zhang
Y
Y
Jacobs
F
F
Martin
L
L
D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D018033:Antibodies, Bispecific; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D004247:DNA; D008668:Metallothionein; D003545:Cysteine