The overall objective of this proposal is to develop a powerful in vitro protein engineering platform that will allow selection or screening of very large numbers of protein variants that is at least 100 -1000 times more cost effective than current 96 well plate based approaches. This strategy is based on generating cell wall bounded micro-compartments, which contain the genetic material, the variant protein and most importantly the product of the enzymatic reaction to allow genotype-phenotype linkage. Using MMLV-reverse transcriptase as a model system, we will develop a novel "permeabilized cell" technology, which is easily adaptable to engineering a broad range of enzymes and proteins and will benefit cancer diagnosis using microarray technology. This technology is a semi in vitro evolution method. The target protein is produced by living cells, which are then permeabilized to allow low molecular weight compounds, such as nucleotides, to diffuse into the cell. The selection step is more like in vitro (within the caged complex formed by the skeleton of fixed cell wall). This technology keeps mutant proteins associated with the DNA encoding them allowing very large numbers of variant proteins to be selected or screened at low cost. PROPOSED COMMERCIAL APPLICATION: Ambion intends to develop an in house protein engineering platform with a novel in vitro evolution methodology. This new technology would provide a powerful tool to evolve proteins with novel functions, such as mutant MMLV-RT and T7 RNA polymerase to incorporate modified nucleotide, which will benefit cancer diagnosis using microarray.