Chaperones facilitate heterologous expression of naturally evolved putative de novo proteins

Abstract

Over the past decade, evidence has accumulated that new protein-coding genes can emerge de novo from previously non-coding DNA. Most studies have focused on large-scale computational predictions of de novo protein coding genes across a wide range of organisms. In contrast, experimental data concerning the folding and function of de novo proteins is scarce. This might be due to difficulties in handling de novo proteins in vitro, as most are predicted to be short and disordered. Here we propose a guideline for the effective expression of eukaryotic de novo proteins in Escherichia coli.We used 11 sequences from Drosophila melanogaster and 10 from Homo sapiens, that are predicted de novo proteins from former studies, for heterologous expression. The candidate de novo proteins have varying secondary structure and disorder content. Using multiple combinations of purification tags, E. coli expression strains and chaperone systems, we were able to increase the number of solubly expressed putative de novo proteins from 30 % to 62 %. Our findings indicate that the best combination for expressing putative de novo proteins in E. coli is a GST-tag with T7 Express cells and co-expressed chaperones. We found that, overall, proteins with higher predicted disorder were easier to express.

The permissive binding theory of cancer

“…The second error is more direct: even random sequences do bind cellular proteins. This seems surprising even to me, but I have been convinced by several recent studies showing that proteins made from random amino acid sequences can bind to cellular proteins and thereby produce essential cellular functions (27)(28)(29). The implication here is that protein binding is reasonably common at baseline, even without shared domains.…”