(427f) Accelerating the Design of Split Proteins with a Range of Cooperative Functions

Functional analysis of adenylate kinase (AK) orthologs bisected at a single, structurally related site suggest that the activity of split proteins correlates with the thermostability of the protein bisected. To test this, we are creating libraries of randomly split AK orthologs from the mesophile Bacillus subtilis and hyperthermophile Thermotoga neapolitana, and we are examining how protein thermostability influences the fraction of variants that cooperatively function. Libraries of vectors that express bisected variants of each AK are being created through a three step protocol in which: i) Hyper MuA is used to randomly insert a minitransposon having flanking notI restriction sites into AK expression vectors, ii) bisected adk genes are purified and subcloned into a second expression vector, and iii) the minitransposon is replaced by a regulatory element that allows for split protein expression. Bisection of protein orthologs with a spectrum of thermostabilities is expected to accelerate the discovery of protein fragments with a range of cooperative functions. In addition, a comparison of the bisection sites in mesophilic and thermophilic AK orthologs that result in split proteins that complement the growth defects of Escherichia coli and Thermus thermophilus having a temperature-sensitive AK will establish whether a protein can gain extra robustness to polypeptide bisection by increasing its thermodynamic stability.