Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-Cys containing peptides but the requirement of a Cys residue at the ligation junction can limit its use. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

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The activity and localization of PTEN, a tumor suppressor lipid phosphatase that converts the phospholipid PIP3 to PIP2, is governed in part by phosphorylation on a cluster of four Ser and Thr residues near the C terminus. Prior enzymatic characterization of the four monophosphorylated (1p) PTENs by using classical expressed protein ligation (EPL) was complicated by the inclusion of a non‐native Cys at the ligation junction (aa379), which may alter the properties of the semisynthetic protein. Here, we apply subtiligase‐mediated EPL to create wt 1p‐PTENs. These PTENs are more autoinhibited than previously appreciated, consistent with the role of Tyr379 in driving autoinhibition. Alkaline phosphatase sensitivity analysis revealed that these autoinhibited 1p conformations are kinetically labile. In contrast to the Cys mutant 1p‐PTENs, which are poorly recognized by an anti‐phospho‐PTEN antibody, three of the four wt 1p‐PTENs are recognized by a commonly used anti‐phospho‐PTEN antibody.

We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2012 The Protein Society into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement.