Multivariate-activity mining for molecular quasi-species in a glutathione transferase mutant library.

A library of recombinant glutathione transferases (GSTs) generated by shuffling of DNA encoding human GST M1-1 and GST M2-2 was screened with eight alternative substrates, and the activities were subjected to multivariate analysis. Assays were made in lysates of bacteria in which the GST variants had been expressed. The primary data showed clustering of the activities in eight-dimensional substrate-activity space.

Expression and purification of catalytically active human PHD3 in Escherichia coli.

Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets.

Functionally diverging molecular quasi-species evolve by crossing two enzymes.

Molecular evolution is frequently portrayed by structural relationships, but delineation of separate functional species is more elusive. We have generated enzyme variants by stochastic recombinations of DNA encoding two homologous detoxication enzymes, human glutathione transferases M1-1 and M2-2, and explored their catalytic versatilities. Sampled mutants were screened for activities with eight alternative substrates, and the activity fingerprints were subjected to principal component analysis.

Directed enzyme evolution guided by multidimensional analysis of substrate-activity space.

The directed evolution of protein function frequently involves identification of mutants with improved properties from a population of variants obtained by mutagenesis. The selection of clones to parent the subsequent generation is crucial to the continued creation of superior progeny. In the present study, multivariate analysis guided the evolution of human glutathione transferase (GST) T1-1 to 65-fold enhanced alkyltransferase activity.