Dataset Information

Enhanced secretion of human ?1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

ABSTRACT: Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor ?1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)₂₀, was engineered either at the N- or C-terminal end of AAT. The (AP)₂₀ tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)₂₀-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)₂₀ along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)₂₀ module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.

SUBMITTER: Zhang N

PROVIDER: S-EPMC6527068 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

Zhang Ningning N Wright Tristen T Caraway Paige P Xu Jianfeng J

Bioengineered 20191201 1

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)<sub>20</sub>, was engineered either at the N- or C-terminal end of AAT. The (A ...[more]

PMID: 30957636

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Project description:α1-antitrypsin (AAT) deficiency is a common autosomal recessive hereditary disorder, with a high risk for the development of early-onset panacinar emphysema. AAT, produced primarily in the liver, functions to protect the lung from neutrophil protease; with AAT deficiency, unimpeded neutrophil proteases destroy the lung parenchyma. AAT is susceptible to oxidative damage resulting in an inability to inhibit its target proteases, neutrophil elastase, and cathepsin G. The major sites of oxidative modification on the AAT molecule are methionine residues 351 and 358. We have previously demonstrated that an engineered variant of AAT that resists oxidation by modifying both protein surface methionines (M351V and M358L) provides antiprotease protection, despite oxidative stress. In mice, intravenous delivery of the modified AAT(AVL) variant by AAV serotype 8, AAV8hAAT(AVL), primarily to the liver resulted in long-term expression of an AAT that resists oxidative inactivation. In this study, we evaluated the safety of intravenous administration of AAV8hAAT(AVL) in a dose-escalating, blinded, placebo-controlled toxicology study in wild-type mice. The study assessed organ histology and clinical pathology findings of mice, intravenously administered AAV8hAAT(AVL) at three doses (5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 genome copies [gc]/kg), compared to control mice injected intravenously with phosphate-buffered saline. As previously demonstrated, administration of AAV8hAAT(AVL) resulted in dose-dependent expression of high, potentially therapeutic, levels of serum human AAT protein that persist for at least 6 months. Antibodies against the AAV8 capsid were elicited as expected, but there was no antibody detected against the AAT(AVL) protein generated by the AAV8hAAT(AVL) vector. There was no morbidity or mortality observed in the study. The data demonstrate that intravenous administration of AAV8hAAT(AVL) is safe with no significant adverse effect attributed to AAV8hAAT(AVL) vector at any dose. This study demonstrates that AAV8hAAT(AVL) has a safety profile consistent with the requirements for proceeding to a clinical study.

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Dataset Information

Enhanced secretion of human ?1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

Publications

Enhanced secretion of human α1-antitrypsin expressed with a novel glycosylation module in tobacco BY-2 cell culture.

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