Expression and Purification of Functional Human α-1-Antitrypsin from Cultured Plant Cells

Huang, Jianmin; Sutliff, Thomas D.; Wu, Liying; Nandi, Somen; Benge, Kelli; Terashima, Masaaki; Ralston, Annemarie H.; Drohan, William N.; Huang, Ning; Rodriguez, Raymond L.

doi:10.1021/bp0001516

Cited by 109 publications

(91 citation statements)

References 23 publications

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“…In the lanes showing the complex, another smaller immunoreactive protein is observed. This observation is consistent with earlier reports that the reaction between a1-PI and elastase results in the cleavage of a1-PI, leaving a smaller portion unattached (Huang et al, 2001;Travis et al, 1985).…”

Section: Characterization Of the A1-pi Variantssupporting

confidence: 93%

“…a1-PI rapidly inactivates neutrophil elastase and therefore reduced tissue proteolysis. a1-PI deficiency can result in lung emphysema in adults, liver disease in children and is also associated with cystic fibrosis, arthritis and other malignant conditions (Huang et al, 2001;Mattes et al, 2001;Travis et al, 1985). Human plasmaderived a1-PI is a FDA----licensed product used for replacement therapy.…”

Section: Introductionmentioning

confidence: 99%

“…Significant research efforts were directed to produce recombinant human a1-PI in E. coli (Courtney et al, 1984), yeast (Kwon et al, 1995), transgenic mice (Zbikowska et al, 2002) and sheep (Wright et al, 1991) as well as plant cells (Huang et al, 2001). E. coli produced an unglycosylated product, the yeast produced incorrect glycosylation, and the transgenic animals pose the same threats as plasma derived product.…”

Section: Introductionmentioning

confidence: 99%

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Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Chill

Trinh

Azadi

et al. 2008

Biotech & Bioengineering

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Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

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Section: Characterization Of the A1-pi Variantssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Chill

Trinh

Azadi

et al. 2008

Biotech & Bioengineering

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“…Cleavage of the RCL at 354 E has been found to be typical of the lysosomal Cys proteinase cathepsin L (Johnson et al, 1986) and nontarget Ser proteases (Nelson et al, 1998). Considerable amounts of truncated protein were also obtained in previous attempts to express recombinant A1AT in rice (Oryza sativa) cells (Terashima et al, 1999;Huang et al, 2001;Trexler et al, 2002;McDonald et al, 2005), N. benthamiana (Sudarshana et al, 2006), and Nicotiana tabacum chloroplasts (Nadai et al, 2009). Nevertheless, A1AT produced in transgenic tomato (Solanum lycopersicum) plants does not show degradation (Jha et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

Castilho¹,

Windwarder

Gattinger³

et al. 2014

Plant Physiology

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Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor a1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g 21 of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.

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“…We first assessed purified A1AT by Coomassie Blue or silver staining methods and then quantified it using bicinchoninic acid assay (Pierce), followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with specific-A1AT antibodies using Western blot. Eluted fractions were tested for retained A1AT activity in cell-free elastase assays as previously described (12).…”

Section: A1at Purificationmentioning

confidence: 99%

Effect of Cigarette Smoke Exposure and Structural Modifications on the α-1 Antitrypsin Interaction with Caspases

Lockett

Demark

et al. 2012

Mol Med

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α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

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Expression and Purification of Functional Human α-1-Antitrypsin from Cultured Plant Cells

Cited by 109 publications

References 23 publications

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Proteolytic andN-Glycan Processing of Humanα1-Antitrypsin Expressed inNicotiana benthamiana

Effect of Cigarette Smoke Exposure and Structural Modifications on the α-1 Antitrypsin Interaction with Caspases

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