In vivo fragment complementation of a (beta/alpha)(8) barrel protein: generation of variability by recombination.
The high representation of the TIM barrel as a scaffold for enzymatic proteins makes it an interesting model for protein engineering. Based on previous reports of folding mechanisms of TIM barrels that suggest an independent folding unit formed by six (beta/alpha) subunits, we interrupted the gene of phosphoribosylanthranilate isomerase (PRAI) from Escherichia coli at three different positions to yield fragments with different combinations of (beta/alpha) subunits. When these constructions were expressed as polycistrons in a TrpF-E. coli strain, complementation of the function only occurred with fragments beta1-alpha4 and beta5-alpha8, demonstrating that (beta/alpha)(4) subunits are stable enough to survive in vivo conditions and to assemble to yield a functional enzyme. The expression of these fragments in a separated plasmid/phagemid system to complement the function gave a slower complementation in the TrpF-E. coli strain; this was overcome by introducing extra secondary elements to the structure that reinforce their interaction.
Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62271, Mexico.
Xavier
X
Soberón
Patricia
P
Fuentes-Gallego
Gloria
G
Saab-Rincón
D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D019143:Evolution, Molecular; D005796:Genes; D005798:Genes, Bacterial; D005816:Genetic Complementation Test / Q000379:methods; D014644:Genetic Variation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D010446:Peptide Fragments; D014305:Triose-Phosphate Isomerase