Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris

Authors: Naumann, Todd; Sollenberger, Kurtis; Hao, Guixia

Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/25/2022
Publication Date: 3/2/2022
Citation: Naumann, T.A., Sollenberger, K.G., Hao, G. 2022. Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris. Protein Expression and Purification. 194. Article 106076. https://doi.org/10.1016/j.pep.2022.106076.
DOI: https://doi.org/10.1016/j.pep.2022.106076

Interpretive Summary: Fungi that cause corn ear rot and mycotoxin contamination of grain have proteins that help to make them successful pathogens. Proteins are the link between DNA and the physical characteristics of an organism. Determining the three-dimensional structure of key pathogen proteins is crucial to understanding how to prevent disease. One of the most powerful techniques that aids protein structural determination is production of selenium containing proteins in a surrogate organism. We developed an improved method for producing selenium containing proteins using yeast. The new method resulted in production of large amounts of selenium proteins that enable structural determination of a fungal protein that promotes corn ear rot and mycotoxin contamination of grain. Determining the structure of fungal proteins will help plant breeders improve plant health and food safety. The improved method will also help protein scientists determine other protein structures more easily.

Technical Abstract: Producing recombinant proteins with incorporated selenomethionine (SeMet) facilitates solving X-ray crystallographic structures of novel proteins. Production of SeMet labeled proteins in the yeast Pichia pastoris (syn. Komagataella phaffii) is difficult because SeMet is mildly toxic, reducing protein expression levels. To counteract this yield loss for a novel protease, Epicoccum sorghi chitinase modifying protein (Es-cmp), a novel disease promoting protease secreted by these plant pathogenic fungi, we isolated a yeast strain that secreted more protein. By comparing the expression level of 48 strains we isolated one that produced significantly more protein. This strain was found to be gene dosed, having four copies of the expression cassette. After optimization the strain produced Es-cmp in defined media with SeMet at levels nearly equal to that of the original strain in complex media. Also, we produced SeMet labeled protein for a homologous protease from the fungus Fusarium vanettenii, Fvan-cmp, by directly selecting a gene dosed strain on agar plates with increased zeocin. Linearization of plasmid with PmeI before electroporation led to high numbers of 1 mg/mL zeocin resistant clones with significantly increased expression compared to those selected on 0.1 mg/mL. The gene dosed strains expressing Es-cmp and Fvan-cmp allowed production of 8.5 and 16.8 mg of SeMet labeled protein from 500 mL shake flask cultures. The results demonstrate that selection of P. pastoris expression strains by plating after transformation on agar with 1 mg/mL zeocin rather than the standard 0.1 mg/mL directly selects gene dosed strains that can facilitate production of selenomethionine labeled proteins.