Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).
We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.
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Technology Research Group, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. mingyue.he@bbsrc.ac.uk
Taussig
M J
MJ
D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D019992:Databases as Topic; D004800:Enzymes, Immobilized / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008280:Magnetics; D051379:Mice; D008822:Mice, Transgenic; D008863:Microspheres; D016133:Polymerase Chain Reaction; D011374:Progesterone / Q000276:immunology; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004800:Enzymes, Immobilized; D007128:Immunoglobulin Fragments; D011506:Proteins; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D008156:Luciferases