Journal article
Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes
Department of Biotechnology and Biomedicine, Technical University of Denmark1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Enzyme Engineering & Structural Biology, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed.
A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test.
Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising in silico selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification.
We have applied this pipeline to a test set of Arabidopsis thaliana cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble: insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio.
We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from Arabidopsis thaliana, and processed this enzyme to near-purity in unprecedented milligram amounts.The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.
Language: | English |
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Publisher: | Public Library of Science |
Year: | 2017 |
Pages: | e0177591 |
ISSN: | 19326203 |
Types: | Journal article |
DOI: | 10.1371/journal.pone.0177591 |
ORCIDs: | Welner, Ditte Hededam |
Agricultural and Biological Sciences (all) Arabidopsis thaliana Article Biochemistry, Genetics and Molecular Biology (all) Cell Wall Enzyme Activation Gene Expression Glycosyltransferases High-Throughput Screening Assays Medicine Plant Cells Q R Recombinant Proteins Science cell wall chaperone computer model enzyme activation enzyme activity enzyme analysis enzymology gene expression genetics glycosyltransferase heterologous expression high throughput screening isolation and purification metabolism nonhuman plant cell plant cell wall glycosyltransferase protein expression recombinant enzyme recombinant protein reversibly glycosylated polypeptide 1 solubility unclassified drug