Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

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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.
Original languageEnglish
Article number0177591
JournalP L o S One
Volume12
Issue number6
Number of pages20
ISSN1932-6203
DOIs
Publication statusPublished - 2017
CitationsWeb of Science® Times Cited: No match on DOI

    Research areas

  • Medicine, R, Science, Q, Cell Wall, Enzyme Activation, Gene Expression, Glycosyltransferases, High-Throughput Screening Assays, Plant Cells, Recombinant Proteins, Biochemistry, Genetics and Molecular Biology (all), Agricultural and Biological Sciences (all), chaperone, glycosyltransferase, plant cell wall glycosyltransferase, recombinant enzyme, reversibly glycosylated polypeptide 1, unclassified drug, recombinant protein, Arabidopsis thaliana, Article, cell wall, computer model, enzyme activity, enzyme analysis, heterologous expression, high throughput screening, nonhuman, protein expression, solubility, enzyme activation, enzymology, gene expression, genetics, isolation and purification, metabolism, plant cell

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