Highly Diverse Protein Library Based on the Ubiquitous (β/α)₈ Enzyme Fold Yields Well‐Structured Proteins through in Vitro Folding Selection

Abstract

Proper protein folding is a prerequisite for protein stability and enzymatic activity. While directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (β/α)8 barrel fold found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)8 barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed an in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants and then combined them to assemble the final library (1014 DNA sequences). The resulting library was analyzed using the in vitro protease assay and an in vivo GFP-folding assay and contains ~1012 soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)8 barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput method mRNA display with a selection for folding. The resulting (β/α)8 barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)8 fold, and to isolate novel enzymes.

Genetic Code Evolution Investigated through the Synthesis and Characterisation of Proteins from Reduced‐Alphabet Libraries

“…Each completed library represented greater than 2×10 12 unique sequences, as determined by spectrophotometric quantification at 260 nm. To confirm the sequences, the final DNA libraries were digested with BsrDI and BsmI and ligated into a pER13 plasmid that had been modified to introduce these restriction sites into the multiple cloning site . Where necessary, further sequencing was performed on variants cloned into pET28a by using the NcoI and BamHI restriction sites.…”

Advances in the directed evolution of proteins

“…Alternatively, a library maximizing complexity while enriching for well-folded proteins was constructed based on one of nature’s most common enzyme folds, the (β/α) 8 barrel fold. All residues on the catalytic face of the protein scaffold were randomized and, simultaneously, the library was enriched for protease resistance by an mRNA display selection, which has been correlated with well-folded and therefore more likely functional proteins [16•]. …”

Thermostable Artificial Enzyme Isolated by In Vitro Selection

“…Furthermore, the in vitro format of this method allows selecting for activity under a wide range of conditions, which is similar to the common approach of screening much smaller libraries of purified proteins, but in contrast to in vivo selection strategies where maintenance of cell viability limits the experimental possibilities. Previous reports on mRNA display include the improvement of folding and stability of proteins by selecting for resistance to protease degradation [38] , or by selecting in the presence of increasing amounts of the denaturant guanidine hydrochloride [39] , [40] . Interestingly, in parallel to our successful selection for RNA ligases at elevated temperature, we also attempted a similar selection in presence of guanidine hydrochloride, but no enrichment was observed even after six rounds (data not shown).…”