Development of expression-ready constructs for generation of proteomic libraries

Methods Mol Biol. 2011;723:257-72. doi: 10.1007/978-1-61779-043-0_17.

Abstract

We describe a method for high-throughput production of protein expression-ready clones. Open-reading frames (ORFs) are amplified by PCR from sequence-verified cDNA clones and subcloned into an appropriate loxP-containing donor vector. Each ORF is represented by two types of clones, one containing the native stop codon for expression of the native protein or amino-terminal fusion constructs and the other made without the stop codon to allow for carboxy-terminal fusion constructs. The expression-ready clone is sequenced to verify that no PCR errors have been introduced. We have made over 11,000 clones ranging in size from 78-6,699 bp with a median of 1,056 bp. This is the largest set of fully sequence-verified-"movable ORFs" of any model organism genome project. The donor clone facilitates rapid and simple transfer of the ORF into any expression vector of choice. Vectors are available for expressing these ORFs in bacteria, cell lines, or transgenic animals. The flexibility of this ORF clone collection makes possible a variety of proteomic applications, including protein interaction mapping, high-throughput cell-based expression screens, and functional studies. We have transferred 5,800 ORFs to a vector that allows production of a FLAG-HA tagged protein in Drosophila tissue culture cells with a metallothionein-inducible promoter. These clones are being used to produce a protein complex map of Drosophila from Schneider cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ampicillin / pharmacology
  • Bacteria / cytology
  • Bacteria / drug effects
  • Bacteria / genetics
  • Cell Culture Techniques
  • Cell Extracts
  • Chromatography, Gel
  • Cloning, Molecular
  • Computational Biology
  • DNA Primers / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Drug Resistance, Bacterial
  • Electrophoresis, Agar Gel
  • Gene Expression
  • Gene Library*
  • Genetic Vectors / genetics
  • Open Reading Frames / genetics
  • Polymerase Chain Reaction
  • Proteomics / methods*
  • Transformation, Bacterial

Substances

  • Cell Extracts
  • DNA Primers
  • Ampicillin
  • endodeoxyribonuclease DpnI
  • Deoxyribonucleases, Type II Site-Specific