Genetic intolerance analysis as a tool for protein science.

Recent advances in whole genome and exome sequencing have dramatically increased the database of human gene variations. There are now enough sequenced human exomes and genomes to begin to identify gene variations that are notable because they are NOT observed in sequenced human genomes, apparently because they are subject to "purifying selection", exemplifying genetic intolerance. Such "dysprocreative" gene variations are embryonic lethal or prevent reproduction through any one of a number of possible mechanisms. Here we review an emerging quantitative approach, "Missense Tolerance Ratio" (MTR) analysis, that is used to assess protein-encoding gene (cDNA) sequence intolerance to missense mutations based on analysis of the >100 K and growing number of currently available human genome and exome sequences. This approach is already useful for analyzing intolerance to mutations in cDNA segments with a resolution on the order of 90 bases. Moreover, as the number of sequenced genomes/exomes increases by orders of magnitude it may eventually be possible to assess mutational tolerance in a statistically robust manner at or near single site resolution. Here we focus on how cDNA intolerance analysis complements other bioinformatic methods to illuminate structure-folding-function relationships for the encoded proteins. A set of disease-linked membrane proteins is employed to provide examples.

Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system.

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared with the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces up to ∼6-fold full-length proteins. However the majority of polypeptides PURE produces are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ∼2/3rd of that measured using the RTS 100 E. coli HY S30 system. Both of the 2 batch systems suffer from low ribosome recycling efficiency when translating proteins from 82 kD to 224 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination before energy depletion increased Fluc protein yield by ∼1.5 to ∼2-fold, while creatine phosphate and magnesium have synergistic effects when added to the PURE system. Additionally, while adding EF-P to PURE reduced full-length protein translated, it increased the fraction of functional protein and reduced partially translated protein probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.