FUNCTIONAL IMMUNOLIPOSOMES HARBORING A BIOSYNTHETICALLY LIPID-TAGGED SINGLE-CHAIN ANTIBODY

被引:36
作者
LAUKKANEN, ML
ALFTHAN, K
KEINANEN, K
机构
[1] VTT Biotechnology and Food Research, FIN-02044 VTT, Espoo
关键词
D O I
10.1021/bi00204a031
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 [H-3]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 realtime 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.
引用
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页码:11664 / 11670
页数:7
相关论文
共 53 条
[1]  
AHMAD I, 1993, CANCER RES, V53, P1484
[2]  
Barbas Carlos F. Iii, 1993, Current Opinion in Biotechnology, V4, P526, DOI 10.1016/0958-1669(93)90072-5
[3]  
CHOI DS, 1986, J BIOL CHEM, V261, P8953
[4]   RECOMBINANT ANTIBODY METALLOTHIONEIN - DESIGN AND EVALUATION FOR RADIOIMMUNOIMAGING [J].
DAS, C ;
KULKARNI, PV ;
CONSTANTINESCU, A ;
ANTICH, P ;
BLATTNER, FR ;
TUCKER, PW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1992, 89 (20) :9749-9753
[5]   RECOMBINANT COLORIMETRIC ANTIBODIES - CONSTRUCTION AND CHARACTERIZATION OF A BIFUNCTIONAL F(AB)2/ALKALINE PHOSPHATASE CONJUGATE PRODUCED IN ESCHERICHIA-COLI [J].
DUCANCEL, F ;
GILLET, D ;
CARRIER, A ;
LAJEUNESSE, E ;
MENEZ, A ;
BOULAIN, JC .
BIO-TECHNOLOGY, 1993, 11 (05) :601-605
[6]   2-DIMENSIONAL RECOGNITION PATTERN OF LIPID-ANCHORED FAB' FRAGMENTS [J].
EGGER, M ;
HEYN, SP ;
GAUB, HE .
BIOPHYSICAL JOURNAL, 1990, 57 (03) :669-673
[7]  
GENNITY JM, 1991, J BIOL CHEM, V266, P16458
[8]  
GHRAYEB J, 1984, J BIOL CHEM, V259, P463
[9]   LIPOSOMES IN DRUG DELIVERY - CLINICAL, DIAGNOSTIC AND OPHTHALMIC POTENTIAL [J].
GREGORIADIS, G ;
FLORENCE, AT .
DRUGS, 1993, 45 (01) :15-28
[10]  
GREGORIADIS G, 1975, BIOCHEM BIOPH RES CO, V65, P537