CHARACTERIZATION OF A YEAST PARTIAL DE NOVO PROTEIN TO UNDERSTAND NOVEL FOLDING AND EVOLUTION

Aishan Shi , Matthew Cordes and Joanna Masel

CHARACTERIZATION OF A YEAST PARTIAL DE NOVO PROTEIN TO UNDERSTAND NOVEL FOLDING AND EVOLUTION

The prevailing model for gene evolution is that new genes derive from old genes by speciation or duplication. Recently however, a large number of complete de novo genes or gene extensions have been discover that evolved from noncoding DNA. YNRO34W-A is a yeast gene that has two alleles of differing length. The longer sequence results form a frameshift mutation that both alters 33 of 98 amino acids in the shorter sequence, and also extends the sequence by 26 residues. This project aims to understand whether this radical mutation leads to the formation of novel folded structure. We obtained an expression vector for an N-terminally histidine-tagged version of the short allele from the DNASU repository. For the long allele, we obtained a commercially synthesized, codon-optimized version of the gene. Both proteins showed significant expression in E. coli bacteria and could be purified by denaturing nickel affinity chromatography. Dialysis into native buffer led to a high degree of precipitation for the longer allele, hindering efforts to compare its structure to that of the short allele by circular dichroism.  This precipitation may result from misfolding and/or a highly aggregation prone region in the C-terminal extension of the long allele. Efforts are currently underway to purify the proteins by native methods as an alternative. This research is funded by the Arnold and Mabel Beckman Foundation and NIH (grant #313220).

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