High-throughput, fluorescence-based screening for soluble protein expression

Matthew A Coleman, V. H. Lao, B. W. Segelke, P. T. Beernink

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

Original languageEnglish (US)
Pages (from-to)1024-1032
Number of pages9
JournalJournal of Proteome Research
Volume3
Issue number5
DOIs
StatePublished - Sep 2004
Externally publishedYes

Fingerprint

Screening
Fluorescence
Throughput
Proteins
Green Fluorescent Proteins
Transcription
Microarrays
Escherichia coli
Fusion reactions
Protein Array Analysis
Fluorophores
Gene Library
Proteomics
Lysine
Lasers
Gels
Complementary DNA
Genes
Gene Expression
Amino Acids

Keywords

  • BODIPY-FL
  • Fluorescence
  • Green fluorescent protein (GFP)
  • High-throughput screening
  • Protein expression
  • Protein microarray

ASJC Scopus subject areas

  • Genetics
  • Biotechnology
  • Biochemistry

Cite this

High-throughput, fluorescence-based screening for soluble protein expression. / Coleman, Matthew A; Lao, V. H.; Segelke, B. W.; Beernink, P. T.

In: Journal of Proteome Research, Vol. 3, No. 5, 09.2004, p. 1024-1032.

Research output: Contribution to journalArticle

Coleman, Matthew A ; Lao, V. H. ; Segelke, B. W. ; Beernink, P. T. / High-throughput, fluorescence-based screening for soluble protein expression. In: Journal of Proteome Research. 2004 ; Vol. 3, No. 5. pp. 1024-1032.
@article{4e1afe8ab9e54668b71163becb68a965,
title = "High-throughput, fluorescence-based screening for soluble protein expression",
abstract = "Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.",
keywords = "BODIPY-FL, Fluorescence, Green fluorescent protein (GFP), High-throughput screening, Protein expression, Protein microarray",
author = "Coleman, {Matthew A} and Lao, {V. H.} and Segelke, {B. W.} and Beernink, {P. T.}",
year = "2004",
month = "9",
doi = "10.1021/pr049912g",
language = "English (US)",
volume = "3",
pages = "1024--1032",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "5",

}

TY - JOUR

T1 - High-throughput, fluorescence-based screening for soluble protein expression

AU - Coleman, Matthew A

AU - Lao, V. H.

AU - Segelke, B. W.

AU - Beernink, P. T.

PY - 2004/9

Y1 - 2004/9

N2 - Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

AB - Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.

KW - BODIPY-FL

KW - Fluorescence

KW - Green fluorescent protein (GFP)

KW - High-throughput screening

KW - Protein expression

KW - Protein microarray

UR - http://www.scopus.com/inward/record.url?scp=7044235834&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=7044235834&partnerID=8YFLogxK

U2 - 10.1021/pr049912g

DO - 10.1021/pr049912g

M3 - Article

C2 - 15473692

AN - SCOPUS:7044235834

VL - 3

SP - 1024

EP - 1032

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 5

ER -