Lessons from an α-Helical Membrane Enzyme: Expression, Purification, and Detergent Optimization for Biophysical and Structural Characterization

Johnson, Jennifer; KALYONCU UZUNLAR, SİBEL; Lieberman, Raquel

doi:10.1007/978-1-4939-3637-3_18

Lessons from an α-Helical Membrane Enzyme: Expression, Purification, and Detergent Optimization for Biophysical and Structural Characterization

Johnson J. L., KALYONCU UZUNLAR S., Lieberman R. L.

HETEROLOGOUS EXPRESSION OF MEMBRANE PROTEINS: METHODS AND PROTOCOLS, 2ND EDITION, cilt.1432, ss.281-301, 2016 (SCI-Expanded, Scopus)

Yayın Türü: Makale / Tam Makale
Cilt numarası: 1432
Basım Tarihi: 2016
Doi Numarası: 10.1007/978-1-4939-3637-3_18
Dergi Adı: HETEROLOGOUS EXPRESSION OF MEMBRANE PROTEINS: METHODS AND PROTOCOLS, 2ND EDITION
Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
Sayfa Sayıları: ss.281-301
Dokuz Eylül Üniversitesi Adresli: Hayır

Özet

This chapter outlines the protocol developed in our lab to produce a multipass alpha-helical membrane protein. We present our work flow, from ortholog selection to protein purification, including molecular biology for plasmid construction, protein expression in E. coli, membrane isolation and detergent solubilization, protein purification and tag removal, biophysical assessment of protein stability in different detergents, and detergent concentration determination using thin-layer chromatography. We focus on results from our ongoing work with intramembrane aspartyl proteases from archaeal organisms.