A native-like artificial protein from antisense DNA.
We describe the creation of folded chimaeric proteins by combining a designed polypeptide segment (bait) derived from a beta-sheet of a human antibody variable domain with random polypeptide segments encoded by human cDNA fragments. The repertoire of polypeptides was displayed on the surface of filamentous bacteriophage and folded polypeptides were selected by proteolysis. One of these, 2a6, was readily expressed in the Escherichia coli cytoplasm as a soluble and protease-resistant protein and could be purified after heating the bacterial lysate to 90 degrees C. Soluble 2a6 is dimeric and its CD spectrum is consistent with components of both alpha and beta structure. 2a6 cooperatively and reversibly unfolds by heat or urea with a folding energy of 11.4 kcal mol(-1) for the transition between folded dimer and unfolded monomer and its refolding steps proceed without the formation of detectable aggregates. Its stability and folding properties are therefore typical of native proteins. Sequence analysis revealed that the cDNA segment in 2a6 was recruited from the antisense strand of a human gene, suggesting that antisense sequences can provide a reservoir for the evolution of soluble and stable proteins.
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. nfischer@novimmune.com
Nicolas
N
Fischer
Lutz
L
Riechmann
Greg
G
Winter
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000737:chemistry / Q000494:pharmacology
D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D016376:Oligonucleotides, Antisense; D010455:Peptides; D012333:RNA, Messenger; D014508:Urea; D004247:DNA