A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs.
Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.
Nakayama
Manabu
M
Department of Human Gene Research, Kazusa DNA Research Institute, Chiba, Japan. nmanabu@kazusa.or.jp
Ohara
Osamu
O
D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D040081:Protein Array Analysis / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry*; D012995:Solubility
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins