Engineering proteins, subcloning and hyperexpressing oxidoreductase genes

A very efficient system for subcloning and studying proteinsequences, combining previously established elements for hyperexpression,replication and screening, was used to hyperproduce and characterizeseven different products. It expedited the cloning of genes,in a multipurpose recombinant DNA construct, for all the requirementsto study and engineer proteins with a strain of Escherichiacoli. Genes encoding six heme proteins and a flavoprotein havebeen subcloned and expressed to 13–30% of the total cellprotein, greatly facilitating purification and analyses. Threeof the heme proteins and the flavoprotein incorporated prostheticgroups in E.coli, and exhibited the expected activities. Fourof the enzymes have been purified to homogeneity and two ofthese crystallized for X-ray diffraction analysis. A rapid muta-genesisprotocol, based on polymerase chain reactions, was successfullyapplied to clone derivatives of one of these enzymes, cytochromec peroxidase. Thus, this system fulfills all criteria for engineeringproteins in an efficient and concerted manner.