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. 2011 Dec 30;11:128.
doi: 10.1186/1472-6750-11-128.

Antibody Degradation in Tobacco Plants: A Predominantly Apoplastic Process

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Free PMC article

Antibody Degradation in Tobacco Plants: A Predominantly Apoplastic Process

Verena K Hehle et al. BMC Biotechnol. .
Free PMC article

Abstract

Background: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem.

Results: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space.

Conclusions: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

Figures

Figure 1
Figure 1
Western blot analysis of transgenic N. tabacum plants expressing MAb 2G12. Leaf discs from 2G12-expressing plants were extracted in either PBS (-) or PBS supplemented with protease inhibitor cocktail (+) and incubated at room temperature for 24 hours. Proteins were separated by 4-15% SDS-PAGE under non-reducing conditions. The proteins were blotted to nitrocellulose membranes and probed with either anti-human IgG Fc-specific antiserum (Panel A) or anti-human κ antiserum (Panel B). The positive control (PC) consists of the human IgG1κ (1 ng/lane). The asterisk indicates the fully assembled 2G12 antibody; lower case letters (a to i) indicate antibody fragments.
Figure 2
Figure 2
Analysis, under reducing conditions, of purified 2G12. 2G12 and antibody fragments from plant extracts were purified using Protein A/G affinity chromatography. Samples were reduced and separated by SDS-PAGE. In Panel A, separated proteins (15% gel) were detected with Coomassie staining. The positive control (PC) consists of 500 ng/lane of human IgG1κ. The positions of free heavy and light chains are indicated by H and L, respectively. In Panels B and C, separated proteins (4-15% gradient gels) were blotted to nitrocellulose membranes and probed with either human Fc-specific antiserum (Panel B) or anti-kappa antiserum (Panel C). The positions of the free heavy (H) and light (L) chains are indicated, while the lower molecular weight species labelled a to e indicate antibody fragments.
Figure 3
Figure 3
Western blot analysis of exogenous human IgG1κ incubated with wild type N. tabacum leaf extract with or without protease inhibitor cocktail. Leaf discs from wild type N. tabacum plants were extracted in either PBS (-) or PBS supplemented with a protease inhibitor cocktail (+) and exogenous human IgG1κ (1.8 ng/lane) was added. The mixtures were incubated at room temperature for the indicated times and proteins separated by 6% SDS-PAGE under non-reducing conditions. The proteins were blotted onto nitrocellulose and probed with anti-human γ1 (Fc region specific) antiserum (Panel A) or anti-human κ antiserum (Panel B). The control (PC), is a human IgG1κ (1.8 ng/lane). The asterisk indicates the fully assembled 2G12 antibody; lower case letters (a, b and c) indicate antibody fragments.
Figure 4
Figure 4
Western blot analysis of protease-activity in wild type N. tabacum leaf extracts of different pH. Human IgG1κ was mixed with wild-type N. tabacum leaf extracts buffered at pH 3.0, 4.2, 5.0, 6.2, 7.4 or 8.0 and incubated at room temperature for 15 mins (Panel A); 2 hrs (Panel B); or 24 hrs (Panel C). Proteins were separated by 6% SDS-PAGE under non-reducing conditions. The proteins were blotted onto nitrocellulose and probed with anti-human γ1 (Fc region specific) antiserum. PC is the positive control human IgG1κ (1.8 ng/lane). For three of the pH conditions, the effect of a protease inhibitor cocktail was tested (± PI, Panels D and E). Samples were tested at 24 hrs and immunodetection was with anti-heavy chain antiserum (Panel D) or anti-light chain antiserum (Panel E). The asterisk indicates the fully assembled 2G12 antibody; lower case letters (a, b and c) indicate antibody fragments.
Figure 5
Figure 5
Western blot analysis of recombinant MAb 2G12 extracted from transgenic N. tabacum in different pH-buffers. Leaf discs from MAb 2G12-expressing plants were extracted in solutions buffered to; pH 3.0, pH 4.2, pH 5.0, pH 6.2, pH 7.4, or pH 8.0. The mixtures were incubated at room temperature for 15 mins (Panel A) or 24 hrs (Panel B). Proteins were separated by SDS-PAGE under non-reducing conditions. Proteins were blotted onto nitrocellulose membranes and probed with anti-human κ antiserum. The asterisk corresponds to the fully assembled 2G12 antibody, while the lower case letters (a to j) indicate antibody fragments.
Figure 6
Figure 6
Pulse chase analysis of antibody fragments in MAb 2G12 transgenic plant cells. Protoplasts from leaves of transgenic tobacco plants expressing the MAb 2G12 were washed and analysed by SDS-PAGE and western blotting (Panels C and D) or pulse-labelled for 1 hr and chased for the indicated periods of time (Panels A and B). Extracellular medium samples and homogenised cells were subjected to immunoprecipitation with Protein G/Protein A mix. Immunoprecipitated proteins were analyzed by SDS-PAGE and radiography. The asterisk corresponds to the fully assembled 2G12 antibody, while the lower case letters (a to i) indicate antibody fragments. Panels A and C were performed under non-reducing conditions, whereas samples in Panels B and D were reduced with the addition of 5% β-mercaptoethanol prior to boiling.

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