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FEBS Open Bio. 2018 Sep 17;8(10):1711-1721. doi: 10.1002/2211-5463.12515. eCollection 2018 Oct.

Oxidation-resistant and thermostable forms of alpha-1 antitrypsin from Escherichia coli inclusion bodies.

Author information

1
Key Laboratory for Microorganisms and Biotransformation College of Life Science South-Central University for Nationalities Wuhan China.
2
State Key Laboratory of Protein and Plant Gene Research School of Life Sciences Peking University Beijing China.
3
Key Engineering Research Centers of Marine Organisms Medical Products Food and Medicine School of Zhejiang Ocean University Zhoushan China.
4
Marine Fisheries Research Institute of Zhejiang Province Zhoushan China.
5
Cardinal Intellectual Property LLC Oakland CA USA.
6
Shenzhen YHLO Biotech Co., Ltd. Shenzhen China.
7
Wuxi Biortus Biosciences Co., Ltd. Jiangyin China.
8
School of Software Huazhong University of Science and Technology Wuhan China.
9
State Key Laboratory of Medicinal Chemical Biology Nankai University College of Pharmacy Tianjin China.

Abstract

Native α1-antitrypsin (AAT) is a 52-kDa glycoprotein that acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases. The main function of AAT is to protect the lung from proteolytic damage induced by inflammation. AAT deficiency (AATD) is a codominant autosomal disorder caused by pathogenic mutations in SERPINA1 gene, leading to reduced levels of serum AAT. The deficiency is known to increase the risk of pulmonary emphysema and chronic obstructive pulmonary disease as a consequence of proteolytic imbalance induced by inflammation, associated in many instances with cigarette smoking and other environmental hazards. Currently, the available therapy for lung disease associated with AATD is serum purified human AAT injected into patients on a weekly basis. It would be advantageous to replace serum-derived AAT with a recombinant version which is stable and resistant to oxidation. We have expressed AAT in Escherichia coli as inclusion bodies and developed a highly efficient refolding and purification process. We engineered a series of mutant forms of AAT to achieve enhance thermostability and oxidation resistance. Moreover, we synthesized an active form of AAT via cysteine-pegylation to achieve a markedly extended half-life in vivo. The resulting molecule, which retains comparable activity to the wild-type form, is expected to be an improved therapeutic agent for treating hereditary emphysema. In addition, the molecule may also be used to treat other types of emphysema caused by smoking, cystic fibrosis, pulmonary hypertension, pulmonary fibrosis, and chronic obstructive pulmonary disease.

KEYWORDS:

Escherichia coli inclusion body refolding; mutant alpha‐1 antitrypsin; protease inhibitor; protein therapeutic; recombinant protein

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