Engineering proteins, subcloning and hyperexpressing oxidoreductase genes.
A very efficient system for subcloning and studying protein sequences, combining previously established elements for hyperexpression, replication and screening, was used to hyperproduce and characterize seven different products. It expedited the cloning of genes, in a multipurpose recombinant DNA construct, for all the requirements to study and engineer proteins with a strain of Escherichia coli. Genes encoding six heme proteins and a flavoprotein have been subcloned and expressed to 13-30% of the total cell protein, greatly facilitating purification and analyses. Three of the heme proteins and the flavoprotein incorporated prosthetic groups in E. coli, and exhibited the expected activities. Four of the enzymes have been purified to homogeneity and two of these crystallized for X-ray diffraction analysis. A rapid mutagenesis protocol, based on polymerase chain reactions, was successfully applied to clone derivatives of one of these enzymes, cytochrome c peroxidase. Thus, this system fulfills all criteria for engineering proteins in an efficient and concerted manner.
Darwish
K
K
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, Rockville.
Li
H Y
HY
Poulos
T L
TL
D001426:Bacterial Proteins; D003001:Cloning, Molecular / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D003578:Cytochrome-c Peroxidase / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D009211:Myoglobin / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase; D010088:Oxidoreductases / Q000235:genetics*; D010957:Plasmids; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005415:Flavins; D009211:Myoglobin; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D003578:Cytochrome-c Peroxidase; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases