title: Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli.
abstract: This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-D-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland 0745, New Zealand.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Cridge
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland 0745, New Zealand.
authors.forename: Jyothsna
authors.initials: J
authors.lastname: Visweswaraiah
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland 0745, New Zealand.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Ramesh
authors.affiliation: Institute of Natural and Mathematical Sciences, Massey University, Auckland 0745, New Zealand. Electronic address: e.sattlegger@massey.ac.nz.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Sattlegger
mesh_terms: D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering; D007118:Immunoassay / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins
keywords: Colony Western; Colony expression screening; Colony immunoblotting; Escherichia coli; Protein overexpression; Saccharomyces cerevisiae