Identifying Stable Fragments of Arabidopsis thaliana Cellulose Synthase Subunit 3 by Yeast Display

• Published in: Biotechnology journal 2019-04, Vol.14 (4), p.e1800353-n/a
• Main Authors: Raeeszadeh‐Sarmazdeh, Maryam, Patel, Nikhil, Cruise, Sarah, Owen, Leila, O'Neill, Hugh, Boder, Eric T.
• Format: Article
• Language: English
• Subjects: Arabidopsis - enzymology; Arabidopsis - genetics; Cell Membrane - chemistry; Cell Membrane - genetics; Cell Wall - enzymology; Cell Wall - genetics; Cellulose - chemistry; cellulose synthase; domain identification; Gene Expression Regulation, Enzymologic; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; library of random protein fragments; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; protein stability; Saccharomyces cerevisiae - genetics; yeast surface display
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201800353

Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown. Here, yeast surface display (YSD) is used to determine the full‐length expression of A. thaliana cellulose synthase 3 (AtCesA3) fragments. The level of stably‐folded AtCesA3 fragments displayed on the yeast surface are determined using flow cytometric analysis of differential surface expression of epitopes flanking the AtCesA3 fragment. This technique provides a fast and simple method for examining folding and expression of protein domains and fragments of complex proteins. Yeast surface display (YSD) is used to determine full‐length expression of Arabidopsis thaliana cellulose synthase 3 (AtCesA3) fragments. A library of random AtCesA3 fragments displayed on the yeast surface is screened based on full‐length expression level using fluorescent‐activated cell sorting (FACS). This technique provides a fast and simple method for screening protein domains and fragments of complex proteins to identify potential stable protein fragments.