A Human cDNA Expression Library in Yeast Enriched for Open Reading Frames

Abstract

We developed a high-throughput technique for the generation of cDNA libraries in the yeast Saccharomyces cerevisiae which enables the selection of cloned cDNA inserts containing open reading frames (ORFs). For direct screening of random-primed cDNA libraries, we have constructed a yeast shuttle/expression vector, the so-called ORF vector pYEXTSH3, which allows the enriched growth of protein expression clones. The selection system is based on the HIS3 marker gene fused to the C terminus of the cDNA insert. The cDNAs cloned in-frame result in histidine prototrophic yeast cells growing on minimal medium, whereas clones bearing the vector without insert or out-of-frame inserts should not grow on this medium. A randomly primed cDNA library from human fetal brain tissue was cloned in this novel vector, and using robot technology the selected clones were arrayed in microtiter plates and were analyzed by sequencing and for protein expression. In the constructed cDNA expression library, about 60% of clones bear an insert in the correct reading frame. In comparison to unselected libraries it was possible to increase the clones with inserts in the correct reading frame more than fourfold, from 14% to 60%. With the expression system described here, we could avoid time-consuming and costly techniques for identification of clones expressing protein by using antibody screening on high-density filters and subsequently rearraying the selected clones in a new "daughter" library. The advantage of this ORF vector is that, in a one-step screening procedure, it allows the generation of expression libraries enriched for clones with correct reading frames as sources of recombinant proteins.

Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring

“…However, phosphorylated or glycosylated PSAQ standards can be produced using specific production systems (11,13). Certainly, the throughput of PSAQ standard production could be further enhanced by availing of cDNA libraries specifically developed for protein expression (29,30).…”

Protein–Protein Interactions Between Large Proteins: Two-Hybrid Screening Using a Functionally Classified Library Composed of Long cDNAs

“…1B). Although one report describes a yeast expression vector (bearing in-frame cloned ORFs) that can be selected in enrichment media (Holz et al 2001), selection with a yield of >60% for ORF clones with correct reading frames is still difficult to achieve. Moreover, bias arising from such preselection enrichment techniques cannot be avoided.…”

Open Reading Frame Filtering Methods for Identification of Genic Sequences

“…More importantly, ORF filtered library using bacteria provides faster growth rate and higher transformation efficiency [12]. Yeast ORF filtered library is not yet extensively tested as bacteria, Holz C et al, applied ORF filtering on yeast were they achieved 60% ORFs, using insert of 200-20000bp [32,33]. However, given that yeast generally have much lower transformation efficiencies, they are unlikely to be an ideal host except under specific conditions, such as if a fragment is not believed to fold properly except in a eukaryotic environment.…”

“…ORFs have also been tested in yeast, by transforming first into a bacterial strain for expression of the desired vector, which must be ampicillin resistance by plating into Amp plates [32]. Following the selection of the desired vector, the plasmid was transformed into yeast to get ORFs through histidine induction medium to select ORFs that are tagged with histidine gene.…”