Abstract
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.
| Original language | English |
|---|---|
| Article number | 1800353 |
| Journal | Biotechnology Journal |
| Volume | 14 |
| Issue number | 4 |
| DOIs | |
| State | Published - Apr 2019 |
Funding
The authors thank Nancy Nielson at cell sorting facility at UT Institute for Agriculture for the help with Fluorescence-Activated Cell Sorting and the staff of the sequencing facility of the UT Genomics Core for DNA sequencing assistance. Funding was provided by the UTK Sustainable Energy Education and Research Center and a Joint-Directed Research and Development award from the UT-ORNL Science Alliance. H.O’N acknowledges support of the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UT-Batelle, LLC, for the U. S. CSC, cellulose synthase complex; YSD, yeast surface display; FACS, fluorescent-activated cell sorting; AtCesA3, Arabidopsis thaliana cellulose synthase 3. The authors thank Nancy Nielson at cell sorting facility at UT Institute for Agriculture for the help with Fluorescence-Activated Cell Sorting and the staff of the sequencing facility of the UT Genomics Core for DNA sequencing assistance. Funding was provided by the UTK Sustainable Energy Education and Research Center and a Joint-Directed Research and Development award from the UT-ORNL Science Alliance. H.O'N acknowledges support of the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UT-Batelle, LLC, for the U. S. Department of Energy (DOE) under contract No. DE-AC05-00OR22725 and the Center for Lignocellulose Structure and Formation (CLSF), an Energy Frontier Research Center funded by the U.S. DOE, Office of Science, Office of Basic Energy Sciences. The authors declare no commercial or financial conflict of interest. Department of Energy (DOE) under contract No. DE-AC05-00OR22725 and the Center for Lignocellulose Structure and Formation (CLSF), an Energy FrontierResearchCenterfundedbytheU.S.DOE,OfficeofScience,Officeof Basic Energy Sciences.
| Funders | Funder number |
|---|---|
| Center for Lignocellulose Structure and Formation | |
| U. S. | |
| UT-Batelle | |
| UT-ORNL Science Alliance | |
| UTK Sustainable Energy Education and Research Center | |
| U.S. Department of Energy | DE-AC05-00OR22725 |
| Office of Science | |
| Basic Energy Sciences | |
| Oak Ridge National Laboratory |
Keywords
- cellulose synthase
- domain identification
- library of random protein fragments
- protein stability
- yeast surface display