In vivo fragment complementation of a (β/α)8 barrel protein: generation of variability by recombination
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- 作者：Soberó ; n ; Xavier ; Fuentes-Gallego ; Patricia ; Saab-Rincó ; n ; Gloria
- 关键词：TIM barrel ; Fragment complementation ; Directed evolution ; Phosphoribosylanthranilate isomerase ; Protein engineering
- 刊名：FEBS Letters
- 出版年：2004
- 出版时间：February 27, 2004
- 年：2004
- 卷：560
- 期：1-3
- 页码：167-172
- 全文大小：424 K
文摘
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 (β/α) subunits, we interrupted the gene of phosphoribosylanthranilate isomerase (PRAI) from Escherichia coli at three different positions to yield fragments with different combinations of (β/α) subunits. When these constructions were expressed as polycistrons in a TrpF-E. coli strain, complementation of the function only occurred with fragments β1-α4 and β5-α8, demonstrating that (β/α)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.