Boehringer Mannheim Gmbh

Mark Krevolin

Scott J Eisenbeis

David J Ledden

Sophie J Boguslawski

Scott Clark

Christopher P Bryant

Muteins of enzyme acceptor polypeptide fragments of .beta.-galactosidase are provided which exhibit substantially increased kinetic complementation activity with no significant loss in stability. A preferred enzyme acceptor fragment has an amino acid other than cysteine located at position 500 of the natural sequence. An especially preferred substitution is serine or valine. Other preferred muteins have an amino acid other than methionine located at position 443, with leucine being especially preferred, or an amino acid other than cysteine at position 76, with leucine being an especially preferred substitution. Also provided are methods for producing the novel muteins, reagent compositions comprising the novel muteins, and immunoassay methods for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

Muteins of .beta.-galactosidase fragments having increased activity

A mutein of an enzyme acceptor polypeptide fragment of .beta.-galactosidase which is resistant to oxidation is provided. The enzyme acceptor fragment has an amino acid other than cysteine located at position 602 of the natural sequence. An especially preferred substitution is serine. Also provided are a method for producing the novel mutein, a reagent composition comprising the novel mutein, and an immunoassay method for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

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Pinole, CA, United States of America

Mark Krevolin