Functional immunoliposomes harboring a biosynthetically lipid-tagged single-chain antibody.
An anti-2-phenyloxazolone single-chain antibody was expressed in Escherichia coli as a lipoprotein fusion in order to generate a biosynthetically lipid-tagged molecule [Laukkanen et al. (1993) Protein Eng. 6, 449-454]. For purification, a hexahistidinyl tag was introduced to the C-terminus of the protein. The resulting antibody, termed Ox lpp-scFv-H6, was membrane-bound, displayed hapten-binding activity, and contained the lipoprotein-specific lipid modification as indicated by metabolic [3H]palmitic acid labeling. The Ox lpp-scFv-H6 was purified by immobilized metal affinity chromatography followed by hapten-based affinity chromatography to essential homogeneity with a yield of 0.4-1.6 mg/L of culture. In detergent dialysis, the purified antibody partitioned quantitatively into phospholipid liposomes. The immunoliposome preparation consisting of a homogeneous population of unilamellar 100-200 nm vesicles displayed specific hapten-binding activity as measured by using ELISA and surface plasmon resonance (SPR)-based real-time biospecific interaction analysis. In SPR experiments, the immunoliposomes exhibited virtually irreversible binding to immobilized hapten compared to soluble antibody fragments, consistent with the predicted multivalent binding. Biosynthetic lipid-tagging of antibodies may prove useful for immunoliposome-based diagnostic and therapeutic applications.
Laukkanen
M L
ML
VTT Biotechnology and Food Research, Espoo, Finland.
Alfthan
K
K
Keinänen
K
K
D000906:Antibodies / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D016503:Drug Delivery Systems; D004926:Escherichia coli / Q000235:genetics; D006241:Haptens / Q000276:immunology; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics; D008081:Liposomes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010081:Oxazolone / Q000031:analogs & derivatives / Q000276:immunology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D000906:Antibodies; D006241:Haptens; D008074:Lipoproteins; D008081:Liposomes; D011993:Recombinant Fusion Proteins; C018361:2-phenyloxazolone; D010081:Oxazolone