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Miscellaneous DNA Vector Systems

PORF - pORF-Clone Vector System

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Vectors for improved cloning of open reading frames in yeast

The yeast shuttle/expression vector pORF-CLONE has been specifically developed for an improved and facilitated selection of cloned cDNA inserts containing open reading frames (ORFs) thus allowing an enriched growth of clones expressing authentic polypeptides. This vector system is particulary useful for the development of a a high-throughput technique for the one-step generation of high-quality cDNA libraries in the yeast Saccharomyces cerevisiae and a direct, time-saving screening of random-primed cDNA libraries. In brief, 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.

FEATURES:

  • ideally suited for the construction of cDNA libraries enriched for yeast clones possessing authentic ORFs
  • eukaryotic host enables highly efficient expression of soluble proteins with correct modifications
  • easy affinity purification and detection of recombinant proteins
  • one-step screening procedure

The pORF-CLONE E. coli / S. cerevisiae shuttle/expression vector offers an innovative approach for generating cDNA libraries which are significantly enriched for ORFs and express authentic polypeptides. For examle, 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%. Using the pORF-CLONE expression system, 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 can be avoided. The advantage of this 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.

PACKAGING: pORF-CLONE vector DNA (5ug) plus 5' and 3' sequencing primers