Single-chain antibodies. Bacterial production, biosynthetic lipid tagging and use in the preparation of immunoliposomes

Dissertation

Marja-Leena Laukkanen

Research output: ThesisDissertationCollection of Articles

Abstract

A single-chain antibody (Ox scFv) was constructed by combining the variable domains of the heavy and light chains of an anti-2-phenyloxazolone IgG via a 28-amino acid residue long natural linker peptide derived from a fungal cellulase. The bacterially expressed Ox scFv was correctly processed and secreted to the periplasm of Escherichia coli, and after cell lysis accumulated into the culture medium. By hapten affinity chromatography the single-chain antibody was purified from the culture medium to essential homogeneity with a yield of 1 - 2 mg/l. In comparison with the intact parental antibody, the Ox scFv showed similar affinity to immobilized hapten but was slightly less stable at low pH and high temperatures. Possibilities for stable immobilization of the single-chain antibody to lipid membranes were investigated. In order to circumvent chemical treatment for constructing a membrane-anchored antibody, the advantage of genetic engineering was utilized and the signal peptide with nine N-terminal amino acid residues of the major lipoprotein (lpp) of E.coli was fused to the N-terminus of the Ox scFv. This design subjected the antibody fusion protein (Ox lpp-scFv) to an in vivo enzymatic addition of a glycerolipid moiety to the N-terminal cysteinyl residue of lpp. The Ox lpp-scFv was expressed in E.coli and, unlike its soluble counterpart, it was firmly associated with the bacterial outer membrane and was metabolically labeled with radioactive palmitate, indicating the biosynthetic lipid-tagging. The lipid-tagged antibody solubilized with nonionic detergents displayed hapten-binding properties comparable to those of the soluble Ox scFv. By removal of the detergent in dialysis the Ox lpp-scFv was incorporated into liposomes with retention of binding activity. In order to facilitate purification by immobilized metal affinity chromatography (IMAC), the C-terminal hexahistidinyl tag was engineered into the Ox lpp-scFv. The Ox lpp-scFv-H6 was purified to essential homogeneity by using a two-step chromatographic procedure with a yield of 0.4 - 1.6 mg from cells harvested from 1 l of culture. In the presence of pure phospholipids the purified lipid-tagged antibody was efficiently incorporated to liposomes by dialysis. The resulting immunoliposomes with a homogeneous population of 100 - 200 nm vesicles showed specific hapten-binding activity as measured by ELISA, surface plasmon resonance (SPR) analysis and time-resolved fluoroimmunoassay (TR-FIA). In all experiments, the binding to the immobilized hapten was inhibited in the presence of soluble hapten in a concentration-dependent manner. The multivalent binding nature of the immunoliposomes was observed by the SPR-analysis in the BIAcore system. The potential of the biosynthetically lipid-tagged antibody for the functionalization of europium (Eu)-chelate loaded liposomes was demonstrated. The lipid-tagged antibody was introduced to liposomes by two different methods: by dialysis or by direct adsorption to premade Eu-chelate-loaded liposomes. Both Eu-loaded immunoliposomes were relatively stable when stored at +4 °C. In comparison with Eu-labeled free single-chain antibody, the immunoliposomal reagent with the higher content of Eu-label produced higher signals and showed better sensitivity in TR-FIA. Biosynthetic lipid-tagging in E.coli is an alternative to present chemical in vitro conjugation for converting the otherwise soluble antibody to membrane-anchored form. The resulting lipid-modified antibody is stoichiometrically labeled at the defined location, and thus it can be immobilized into lipid membranes in a stable, oriented and functional manner.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Keinänen, Kari, Supervisor, External person
Award date2 Jun 1995
Place of PublicationEspoo
Publisher
Print ISBNs951-38-4770-5
Publication statusPublished - 1995
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Single-Chain Antibodies
Haptens
Europium
Lipoproteins
Lipids
Antibodies
Liposomes
Escherichia coli
Dialysis
Affinity chromatography
Surface plasmon resonance
Membrane Lipids
Membranes
Detergents
Culture Media
Amino Acids
Genetic engineering
Cellulase
Palmitates
Protein Sorting Signals

Keywords

  • antibodies
  • production
  • Escherichia coli
  • fusion proteins
  • biosynthesis
  • lipoproteins
  • tagging
  • immunoliposomes
  • theses

Cite this

Laukkanen, Marja-Leena. / Single-chain antibodies. Bacterial production, biosynthetic lipid tagging and use in the preparation of immunoliposomes : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1995. 116 p.
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abstract = "A single-chain antibody (Ox scFv) was constructed by combining the variable domains of the heavy and light chains of an anti-2-phenyloxazolone IgG via a 28-amino acid residue long natural linker peptide derived from a fungal cellulase. The bacterially expressed Ox scFv was correctly processed and secreted to the periplasm of Escherichia coli, and after cell lysis accumulated into the culture medium. By hapten affinity chromatography the single-chain antibody was purified from the culture medium to essential homogeneity with a yield of 1 - 2 mg/l. In comparison with the intact parental antibody, the Ox scFv showed similar affinity to immobilized hapten but was slightly less stable at low pH and high temperatures. Possibilities for stable immobilization of the single-chain antibody to lipid membranes were investigated. In order to circumvent chemical treatment for constructing a membrane-anchored antibody, the advantage of genetic engineering was utilized and the signal peptide with nine N-terminal amino acid residues of the major lipoprotein (lpp) of E.coli was fused to the N-terminus of the Ox scFv. This design subjected the antibody fusion protein (Ox lpp-scFv) to an in vivo enzymatic addition of a glycerolipid moiety to the N-terminal cysteinyl residue of lpp. The Ox lpp-scFv was expressed in E.coli and, unlike its soluble counterpart, it was firmly associated with the bacterial outer membrane and was metabolically labeled with radioactive palmitate, indicating the biosynthetic lipid-tagging. The lipid-tagged antibody solubilized with nonionic detergents displayed hapten-binding properties comparable to those of the soluble Ox scFv. By removal of the detergent in dialysis the Ox lpp-scFv was incorporated into liposomes with retention of binding activity. In order to facilitate purification by immobilized metal affinity chromatography (IMAC), the C-terminal hexahistidinyl tag was engineered into the Ox lpp-scFv. The Ox lpp-scFv-H6 was purified to essential homogeneity by using a two-step chromatographic procedure with a yield of 0.4 - 1.6 mg from cells harvested from 1 l of culture. In the presence of pure phospholipids the purified lipid-tagged antibody was efficiently incorporated to liposomes by dialysis. The resulting immunoliposomes with a homogeneous population of 100 - 200 nm vesicles showed specific hapten-binding activity as measured by ELISA, surface plasmon resonance (SPR) analysis and time-resolved fluoroimmunoassay (TR-FIA). In all experiments, the binding to the immobilized hapten was inhibited in the presence of soluble hapten in a concentration-dependent manner. The multivalent binding nature of the immunoliposomes was observed by the SPR-analysis in the BIAcore system. The potential of the biosynthetically lipid-tagged antibody for the functionalization of europium (Eu)-chelate loaded liposomes was demonstrated. The lipid-tagged antibody was introduced to liposomes by two different methods: by dialysis or by direct adsorption to premade Eu-chelate-loaded liposomes. Both Eu-loaded immunoliposomes were relatively stable when stored at +4 °C. In comparison with Eu-labeled free single-chain antibody, the immunoliposomal reagent with the higher content of Eu-label produced higher signals and showed better sensitivity in TR-FIA. Biosynthetic lipid-tagging in E.coli is an alternative to present chemical in vitro conjugation for converting the otherwise soluble antibody to membrane-anchored form. The resulting lipid-modified antibody is stoichiometrically labeled at the defined location, and thus it can be immobilized into lipid membranes in a stable, oriented and functional manner.",
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author = "Marja-Leena Laukkanen",
note = "Project code: B5SU00064",
year = "1995",
language = "English",
isbn = "951-38-4770-5",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "232",
address = "Finland",
school = "University of Helsinki",

}

Single-chain antibodies. Bacterial production, biosynthetic lipid tagging and use in the preparation of immunoliposomes : Dissertation. / Laukkanen, Marja-Leena.

Espoo : VTT Technical Research Centre of Finland, 1995. 116 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Single-chain antibodies. Bacterial production, biosynthetic lipid tagging and use in the preparation of immunoliposomes

T2 - Dissertation

AU - Laukkanen, Marja-Leena

N1 - Project code: B5SU00064

PY - 1995

Y1 - 1995

N2 - A single-chain antibody (Ox scFv) was constructed by combining the variable domains of the heavy and light chains of an anti-2-phenyloxazolone IgG via a 28-amino acid residue long natural linker peptide derived from a fungal cellulase. The bacterially expressed Ox scFv was correctly processed and secreted to the periplasm of Escherichia coli, and after cell lysis accumulated into the culture medium. By hapten affinity chromatography the single-chain antibody was purified from the culture medium to essential homogeneity with a yield of 1 - 2 mg/l. In comparison with the intact parental antibody, the Ox scFv showed similar affinity to immobilized hapten but was slightly less stable at low pH and high temperatures. Possibilities for stable immobilization of the single-chain antibody to lipid membranes were investigated. In order to circumvent chemical treatment for constructing a membrane-anchored antibody, the advantage of genetic engineering was utilized and the signal peptide with nine N-terminal amino acid residues of the major lipoprotein (lpp) of E.coli was fused to the N-terminus of the Ox scFv. This design subjected the antibody fusion protein (Ox lpp-scFv) to an in vivo enzymatic addition of a glycerolipid moiety to the N-terminal cysteinyl residue of lpp. The Ox lpp-scFv was expressed in E.coli and, unlike its soluble counterpart, it was firmly associated with the bacterial outer membrane and was metabolically labeled with radioactive palmitate, indicating the biosynthetic lipid-tagging. The lipid-tagged antibody solubilized with nonionic detergents displayed hapten-binding properties comparable to those of the soluble Ox scFv. By removal of the detergent in dialysis the Ox lpp-scFv was incorporated into liposomes with retention of binding activity. In order to facilitate purification by immobilized metal affinity chromatography (IMAC), the C-terminal hexahistidinyl tag was engineered into the Ox lpp-scFv. The Ox lpp-scFv-H6 was purified to essential homogeneity by using a two-step chromatographic procedure with a yield of 0.4 - 1.6 mg from cells harvested from 1 l of culture. In the presence of pure phospholipids the purified lipid-tagged antibody was efficiently incorporated to liposomes by dialysis. The resulting immunoliposomes with a homogeneous population of 100 - 200 nm vesicles showed specific hapten-binding activity as measured by ELISA, surface plasmon resonance (SPR) analysis and time-resolved fluoroimmunoassay (TR-FIA). In all experiments, the binding to the immobilized hapten was inhibited in the presence of soluble hapten in a concentration-dependent manner. The multivalent binding nature of the immunoliposomes was observed by the SPR-analysis in the BIAcore system. The potential of the biosynthetically lipid-tagged antibody for the functionalization of europium (Eu)-chelate loaded liposomes was demonstrated. The lipid-tagged antibody was introduced to liposomes by two different methods: by dialysis or by direct adsorption to premade Eu-chelate-loaded liposomes. Both Eu-loaded immunoliposomes were relatively stable when stored at +4 °C. In comparison with Eu-labeled free single-chain antibody, the immunoliposomal reagent with the higher content of Eu-label produced higher signals and showed better sensitivity in TR-FIA. Biosynthetic lipid-tagging in E.coli is an alternative to present chemical in vitro conjugation for converting the otherwise soluble antibody to membrane-anchored form. The resulting lipid-modified antibody is stoichiometrically labeled at the defined location, and thus it can be immobilized into lipid membranes in a stable, oriented and functional manner.

AB - A single-chain antibody (Ox scFv) was constructed by combining the variable domains of the heavy and light chains of an anti-2-phenyloxazolone IgG via a 28-amino acid residue long natural linker peptide derived from a fungal cellulase. The bacterially expressed Ox scFv was correctly processed and secreted to the periplasm of Escherichia coli, and after cell lysis accumulated into the culture medium. By hapten affinity chromatography the single-chain antibody was purified from the culture medium to essential homogeneity with a yield of 1 - 2 mg/l. In comparison with the intact parental antibody, the Ox scFv showed similar affinity to immobilized hapten but was slightly less stable at low pH and high temperatures. Possibilities for stable immobilization of the single-chain antibody to lipid membranes were investigated. In order to circumvent chemical treatment for constructing a membrane-anchored antibody, the advantage of genetic engineering was utilized and the signal peptide with nine N-terminal amino acid residues of the major lipoprotein (lpp) of E.coli was fused to the N-terminus of the Ox scFv. This design subjected the antibody fusion protein (Ox lpp-scFv) to an in vivo enzymatic addition of a glycerolipid moiety to the N-terminal cysteinyl residue of lpp. The Ox lpp-scFv was expressed in E.coli and, unlike its soluble counterpart, it was firmly associated with the bacterial outer membrane and was metabolically labeled with radioactive palmitate, indicating the biosynthetic lipid-tagging. The lipid-tagged antibody solubilized with nonionic detergents displayed hapten-binding properties comparable to those of the soluble Ox scFv. By removal of the detergent in dialysis the Ox lpp-scFv was incorporated into liposomes with retention of binding activity. In order to facilitate purification by immobilized metal affinity chromatography (IMAC), the C-terminal hexahistidinyl tag was engineered into the Ox lpp-scFv. The Ox lpp-scFv-H6 was purified to essential homogeneity by using a two-step chromatographic procedure with a yield of 0.4 - 1.6 mg from cells harvested from 1 l of culture. In the presence of pure phospholipids the purified lipid-tagged antibody was efficiently incorporated to liposomes by dialysis. The resulting immunoliposomes with a homogeneous population of 100 - 200 nm vesicles showed specific hapten-binding activity as measured by ELISA, surface plasmon resonance (SPR) analysis and time-resolved fluoroimmunoassay (TR-FIA). In all experiments, the binding to the immobilized hapten was inhibited in the presence of soluble hapten in a concentration-dependent manner. The multivalent binding nature of the immunoliposomes was observed by the SPR-analysis in the BIAcore system. The potential of the biosynthetically lipid-tagged antibody for the functionalization of europium (Eu)-chelate loaded liposomes was demonstrated. The lipid-tagged antibody was introduced to liposomes by two different methods: by dialysis or by direct adsorption to premade Eu-chelate-loaded liposomes. Both Eu-loaded immunoliposomes were relatively stable when stored at +4 °C. In comparison with Eu-labeled free single-chain antibody, the immunoliposomal reagent with the higher content of Eu-label produced higher signals and showed better sensitivity in TR-FIA. Biosynthetic lipid-tagging in E.coli is an alternative to present chemical in vitro conjugation for converting the otherwise soluble antibody to membrane-anchored form. The resulting lipid-modified antibody is stoichiometrically labeled at the defined location, and thus it can be immobilized into lipid membranes in a stable, oriented and functional manner.

KW - antibodies

KW - production

KW - Escherichia coli

KW - fusion proteins

KW - biosynthesis

KW - lipoproteins

KW - tagging

KW - immunoliposomes

KW - theses

M3 - Dissertation

SN - 951-38-4770-5

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -