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Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

Hehle, VK; Paul, MJ; Roberts, VA; van Dolleweerd, CJ; Ma, JK (2015) Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. The FASEB Journal, 30 (4). pp. 1590-1598. ISSN 0892-6638 https://doi.org/10.1096/fj.15-283226
SGUL Authors: Ma, Julian Paul, Mathew John

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Abstract

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

Item Type: Article
Additional Information: © The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distr ibution, and reproduction in any medium, provided the original work is properly cited.
Keywords: antibody engineering, degradation, proteolysis, Biochemistry & Molecular Biology, 0601 Biochemistry And Cell Biology, 0606 Physiology, 1116 Medical Physiology
SGUL Research Institute / Research Centre: Academic Structure > Infection and Immunity Research Institute (INII)
Journal or Publication Title: The FASEB Journal
ISSN: 0892-6638
Language: ENG
Dates:
DateEvent
28 December 2015Published
Publisher License: Creative Commons: Attribution 4.0
Projects:
Project IDFunderFunder ID
AdG 2010 269110European Research Councilhttp://dx.doi.org/10.13039/501100000781
LSAHB-CT-2003-503565European Commissionhttp://dx.doi.org/10.13039/501100000780
FA804European Cooperation in Science and Technologyhttp://dx.doi.org/10.13039/501100000921
WT093092MAWellcome Trusthttp://dx.doi.org/10.13039/100004440
PubMed ID: 26712217
Go to PubMed abstract
URI: https://openaccess.sgul.ac.uk/id/eprint/107592
Publisher's version: https://doi.org/10.1096/fj.15-283226

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