Enzyme-catalyzed expressed protein ligation

Abstract

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.

Analysis of Site‐Specific Phosphorylation of PTEN by Using Enzyme‐Catalyzed Expressed Protein Ligation

“…Subsequently, wild‐type tetraphosphorylated PTEN was created by using enzyme‐catalyzed EPL involving the engineered protein ligase, subtiligase . Subtiligase catalyzes ligation between the C terminus of a protein or a peptide containing a C‐terminal ester or thioester, and the free N terminus of another peptide .…”

“…Subsequently, wild‐type tetraphosphorylated PTEN was created by using enzyme‐catalyzed EPL involving the engineered protein ligase, subtiligase . Subtiligase catalyzes ligation between the C terminus of a protein or a peptide containing a C‐terminal ester or thioester, and the free N terminus of another peptide . Subtiligase can accommodate a wide variety of residues at the ligation junction, and this sequence tolerance was harnessed to create tetraphosphorylated (4p) PTEN without the Y379C mutation.…”

“…Subtiligase catalyzes ligation between the C terminus of a protein or a peptide containing a C‐terminal ester or thioester, and the free N terminus of another peptide . Subtiligase can accommodate a wide variety of residues at the ligation junction, and this sequence tolerance was harnessed to create tetraphosphorylated (4p) PTEN without the Y379C mutation. Analysis of this PTEN showed that it adopted a closed conformation and was less active toward PIP 3 to a much greater degree than was seen with C379 4p PTEN .…”

“…Subtiligase can accommodate a wide variety of residues at the ligation junction, and this sequence tolerance was harnessed to create tetraphosphorylated (4p) PTEN without the Y379C mutation. Analysis of this PTEN showed that it adopted a closed conformation and was less active toward PIP 3 to a much greater degree than was seen with C379 4p PTEN . This difference between C379 and Y379 4p‐PTENs suggests that previous studies with monophosphorylated (1p) PTENs, which incorporated the Y379C mutation, may also not fully capture the effect of tail phosphorylation on PTEN.…”

“…Mammalian PIP3 commonly includes long‐chain fatty acids, C 18 –C 20 , but these phospholipids show low solubility and are commonly studied in vesicles, complicating enzymologic analysis. Six carbon fatty acid di‐C6‐PIP3 is often used as a surrogate for kinetic studies as these phospholipids are more soluble and previous work on PTEN has validated their utility . In the PTEN enzymatic assays, all four monophosphorylated Y‐PTEN forms showed a decrease in catalytic activity relative to truncated recombinant PTEN (t‐PTEN, aa1–378) or non‐phosphorylated full‐length semisynthetic PTEN (n‐Y‐PTEN; Figure B and Table ).…”