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Probing the α-complementing domain of E. coli β-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition by Eini Poussu; Mauno Vihinen; Lars Paulin; Harri Savilahti is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is Probing the α-complementing domain of E. coli β-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition about?

## Abstract Protein structure‐function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the α‐complementing domain 1 of __Escherichia coli__ β‐galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15‐bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (α‐donors) were analyzed for their ability to restore (by α‐complementation) β‐galactosidase activity of the M15 deletion mutant (α‐acceptor), and the data were correlated to the structure of the β‐galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N‐terminus and loop regions most lik

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Author

Eini Poussu; Mauno Vihinen; Lars Paulin; Harri Savilahti

Publisher

John Wiley and Sons; Wiley (John Wiley & Sons); Wiley-Liss Inc; Wiley (ISSN 0887-3585)

Published

2004

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)