General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides.
The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. An easy detection method for a single amino acid change based on this observation was called "GFP-display." Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). the mobility shift is estimated to a certain extent using a new parameter called the "GD value" calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. aokit@hoku-iryo-u.ac.jp
Takashi
T
Aoki
Toshiyuki
T
Tahara
Keiko
K
Satoh
Hiroyoshi
H
Fujino
Hiroyuki
H
Watabe
D019943:Amino Acid Substitution; D030541:Databases, Genetic; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D016296:Mutagenesis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D022702:Receptors, Adrenergic, beta-3 / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013205:Staphylococcal Protein A / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D014508:Urea / Q000737:chemistry; D018631:ras Proteins / Q000032:analysis
D008164:Luminescent Proteins; D010455:Peptides; D022702:Receptors, Adrenergic, beta-3; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D014508:Urea; D018631:ras Proteins