Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin

Anneli Ritala (Corresponding Author), S Leelavathi, Kirsi-Marja Oksman-Caldentey, V S Reddy, Marja-Leena Laukkanen

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk
Original languageEnglish
Pages (from-to)477-487
Number of pages11
JournalTransgenic Research
Volume23
Issue number3
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

lactoglobulins
Lactoglobulins
antibody detection
Hordeum
allergens
Immunoprecipitation
Allergens
Milk
barley
milk
Antibodies
Single-Chain Antibodies
Food Quality
Food Handling
promoter regions
Dairy Products
Glutens
recombinant antibodies
immunoglobulin E
Ubiquitin

Keywords

  • ß-lactoglobulin-specific antibody
  • barley
  • cell culture
  • D1 scFv
  • grain
  • recombinant

Cite this

@article{380842d04f5c46ad9602673f1ce6cfcc,
title = "Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, {\ss}-lactoglobulin",
abstract = "Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for {\ss}-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 {\%} of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk",
keywords = "{\ss}-lactoglobulin-specific antibody, barley, cell culture, D1 scFv, grain, recombinant",
author = "Anneli Ritala and S Leelavathi and Kirsi-Marja Oksman-Caldentey and Reddy, {V S} and Marja-Leena Laukkanen",
year = "2014",
doi = "10.1007/s11248-014-9783-2",
language = "English",
volume = "23",
pages = "477--487",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer",
number = "3",

}

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin. / Ritala, Anneli (Corresponding Author); Leelavathi, S; Oksman-Caldentey, Kirsi-Marja; Reddy, V S; Laukkanen, Marja-Leena.

In: Transgenic Research, Vol. 23, No. 3, 2014, p. 477-487.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin

AU - Ritala, Anneli

AU - Leelavathi, S

AU - Oksman-Caldentey, Kirsi-Marja

AU - Reddy, V S

AU - Laukkanen, Marja-Leena

PY - 2014

Y1 - 2014

N2 - Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk

AB - Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for ß-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8-1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk

KW - ß-lactoglobulin-specific antibody

KW - barley

KW - cell culture

KW - D1 scFv

KW - grain

KW - recombinant

U2 - 10.1007/s11248-014-9783-2

DO - 10.1007/s11248-014-9783-2

M3 - Article

VL - 23

SP - 477

EP - 487

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 3

ER -