Random PCR-based screening for soluble domains using green fluorescent protein.
A novel approach to screen soluble protein domains is presented, by combining tagged random primer polymerase chain reaction (PCR) method and protein-folding assay using green fluorescent protein. Tagged random primer PCR method was used to amplify random DNA fragments from a template cDNA. The PCR products were fused to the green fluorescent protein (GFP) gene. Then solubilities of their translation products were rapidly monitored by the fluorescence of transformed Escherichia coli colonies on plates. We succeeded in cloning four soluble domains from Vav protein using this method. The present method is applicable to all proteins when cDNAs are available.
Kawasaki
M
M
CREST Research Project, Japan Science and Technology Corporation, Kita-ku, Sapporo 060-0812, Japan.
Inagaki
F
F
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D051567:Proto-Oncogene Proteins c-vav; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility
D017931:DNA Primers; D008164:Luminescent Proteins; D015513:Oncogene Proteins; D011506:Proteins; D051567:Proto-Oncogene Proteins c-vav; D011993:Recombinant Fusion Proteins; C496619:Vav1 protein, mouse; D049452:Green Fluorescent Proteins