Flow cytometric screening of cDNA expression libraries for fluorescent proteins.
Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.
Bessette
Paul H
PH
Department of Chemical Engineering, and Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, California 93106, USA.
Daugherty
Patrick S
PS
D000595:Amino Acid Sequence; D000818:Animals; D037421:Anthozoa / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library
D008164:Luminescent Proteins; D019151:Peptide Library