Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Phakawat Chusuth^a, Ponlawoot Raksat^b,c, Supot Hannongbua^d, Kittikhun Wangkanont^e,f,, Puey Ounjai^b,c,, Thanyada Rungrotmongkol^a,g,*

ABSTRACT: Human ?-7 nicotinic acetylcholine receptor (?-7 nAChR) is an ion channel that plays a significant function in neuronal communication and development. Defects of ?-7 nAChR could cause plethora of neurologic and psychiatric impairments such as schizophrenia, Alzheimer?s, and inflammatory disorders. Hence, ?-7 nAChR is an interesting candidate for drug development targeting neurological and inflammatory diseases. Therefore, the ability to obtain significant amount of the ?-7 nAChR extracellular domain is crucial for further biochemical and structural investigations. We constructed a Pichia pastoris yeast strain that secretes the ?-7 nAChR extracellular domain. The recombinant protein was hyperglycosylated and could be deglycosylated with Endoglycosidase H treatment. Crosslinking experiments suggested that the recombinant protein could form oligomeric states, consistent with the known property of the native ?-7 nAChR. The optimized expression yield was 4.9 mg/l.

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^a	Center of Excellence in Biocatalyst and Sustainable Biotechnology, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand
^b	Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400 Thailand
^c	Center of Excellence on Environmental Health and Toxicology (EHT), Office of Higher Education Commission, Ministry of Education, Bangkok Thailand
^d	Computational Chemistry Center of Excellence, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand
^e	Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand
^f	Center of Excellence for Molecular Crop, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand
^g	Program in Bioinformatics and Computational Biology, Graduate School, Chulalongkorn University, Bangkok 10330 Thailand

* Corresponding author, E-mail: kittikhun.w@chula.ac.th, puey.oun@mahidol.edu, thanyada.r@chula.ac.th

Received 16 Feb 2022, Accepted 6 Jun 2022

Expression and purification of human alpha-7 nicotinic acetylcholine receptor extracellular domain in Pichia pastoris

Phakawat Chusutha, Ponlawoot Raksatb,c, Supot Hannongbuad, Kittikhun Wangkanonte,f,*, Puey Ounjaib,c,*, Thanyada Rungrotmongkola,g,*

Phakawat Chusuth^a, Ponlawoot Raksat^b,c, Supot Hannongbua^d, Kittikhun Wangkanont^e,f,, Puey Ounjai^b,c,, Thanyada Rungrotmongkol^a,g,*