New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates

Fu-Tong Liu, Mark Zinnecker, Toshiyuki Hamaoka, David H. Katz

Research output: Contribution to journalArticle

324 Citations (Scopus)

Abstract

Conjugates of small haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inactivating hapten-specific B lymphocytes that bind determinants attached to D-GL. In order to extend the D-GL tolerance system to more complex protein antigens which are directly related to various human diseases, it is necessary to develop techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form. Using hen egg ovalbumin (OVA) as a prototype protein, herein we describe conjugation and purification methods which involve (1) application of a recently developed coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, i.e., reaction of MBS-modified OVA with thiolated D-GL molecules generated in situ from acetyl-S-D-GL by hydroxylamine, and (2) introduction of biotin moieties into D-GL molecules to allow application of an avidin-biotin system for affinity chromatographic purification of conjugates. The reactions and isolations are carried out under mild conditions and in good yields. The conjugate prepared in this way retained a majority of antigenic determinants of OVA and was devoid of any nonconjugated protein, protein dimers, and oligomers which could pose a serious detriment to the effectiveness of tolerance induction. These methods were also applied to the preparation of D-GL conjugates of insulin. The conjugates were characterized by immunoprecipitin reactions and radioimmunoassays. The potential applications of these methods are discussed.

Original languageEnglish (US)
Pages (from-to)690-697
Number of pages8
JournalBiochemistry
Volume18
Issue number4
StatePublished - 1979
Externally publishedYes

Fingerprint

Copolymers
Ovalbumin
Amino Acids
Haptens
Biotin
Proteins
Purification
Hydroxylamine
Molecules
Lymphocytes
Avidin
Oligomers
Dimers
Lysine
Radioimmunoassay
Ovum
Epitopes
Glutamic Acid
Esters
B-Lymphocytes

ASJC Scopus subject areas

  • Biochemistry

Cite this

New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. / Liu, Fu-Tong; Zinnecker, Mark; Hamaoka, Toshiyuki; Katz, David H.

In: Biochemistry, Vol. 18, No. 4, 1979, p. 690-697.

Research output: Contribution to journalArticle

@article{ebe24560e8b84301be30081d007fc364,
title = "New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates",
abstract = "Conjugates of small haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inactivating hapten-specific B lymphocytes that bind determinants attached to D-GL. In order to extend the D-GL tolerance system to more complex protein antigens which are directly related to various human diseases, it is necessary to develop techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form. Using hen egg ovalbumin (OVA) as a prototype protein, herein we describe conjugation and purification methods which involve (1) application of a recently developed coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, i.e., reaction of MBS-modified OVA with thiolated D-GL molecules generated in situ from acetyl-S-D-GL by hydroxylamine, and (2) introduction of biotin moieties into D-GL molecules to allow application of an avidin-biotin system for affinity chromatographic purification of conjugates. The reactions and isolations are carried out under mild conditions and in good yields. The conjugate prepared in this way retained a majority of antigenic determinants of OVA and was devoid of any nonconjugated protein, protein dimers, and oligomers which could pose a serious detriment to the effectiveness of tolerance induction. These methods were also applied to the preparation of D-GL conjugates of insulin. The conjugates were characterized by immunoprecipitin reactions and radioimmunoassays. The potential applications of these methods are discussed.",
author = "Fu-Tong Liu and Mark Zinnecker and Toshiyuki Hamaoka and Katz, {David H.}",
year = "1979",
language = "English (US)",
volume = "18",
pages = "690--697",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "4",

}

TY - JOUR

T1 - New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates

AU - Liu, Fu-Tong

AU - Zinnecker, Mark

AU - Hamaoka, Toshiyuki

AU - Katz, David H.

PY - 1979

Y1 - 1979

N2 - Conjugates of small haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inactivating hapten-specific B lymphocytes that bind determinants attached to D-GL. In order to extend the D-GL tolerance system to more complex protein antigens which are directly related to various human diseases, it is necessary to develop techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form. Using hen egg ovalbumin (OVA) as a prototype protein, herein we describe conjugation and purification methods which involve (1) application of a recently developed coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, i.e., reaction of MBS-modified OVA with thiolated D-GL molecules generated in situ from acetyl-S-D-GL by hydroxylamine, and (2) introduction of biotin moieties into D-GL molecules to allow application of an avidin-biotin system for affinity chromatographic purification of conjugates. The reactions and isolations are carried out under mild conditions and in good yields. The conjugate prepared in this way retained a majority of antigenic determinants of OVA and was devoid of any nonconjugated protein, protein dimers, and oligomers which could pose a serious detriment to the effectiveness of tolerance induction. These methods were also applied to the preparation of D-GL conjugates of insulin. The conjugates were characterized by immunoprecipitin reactions and radioimmunoassays. The potential applications of these methods are discussed.

AB - Conjugates of small haptens and a synthetic copolymer of D-glutamic acid and D-lysine (D-GL) have been shown to be very effective in inactivating hapten-specific B lymphocytes that bind determinants attached to D-GL. In order to extend the D-GL tolerance system to more complex protein antigens which are directly related to various human diseases, it is necessary to develop techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form. Using hen egg ovalbumin (OVA) as a prototype protein, herein we describe conjugation and purification methods which involve (1) application of a recently developed coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, i.e., reaction of MBS-modified OVA with thiolated D-GL molecules generated in situ from acetyl-S-D-GL by hydroxylamine, and (2) introduction of biotin moieties into D-GL molecules to allow application of an avidin-biotin system for affinity chromatographic purification of conjugates. The reactions and isolations are carried out under mild conditions and in good yields. The conjugate prepared in this way retained a majority of antigenic determinants of OVA and was devoid of any nonconjugated protein, protein dimers, and oligomers which could pose a serious detriment to the effectiveness of tolerance induction. These methods were also applied to the preparation of D-GL conjugates of insulin. The conjugates were characterized by immunoprecipitin reactions and radioimmunoassays. The potential applications of these methods are discussed.

UR - http://www.scopus.com/inward/record.url?scp=0018796232&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0018796232&partnerID=8YFLogxK

M3 - Article

VL - 18

SP - 690

EP - 697

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 4

ER -