Method Article
Expressed Protein Ligation using subtiligase
https://doi.org/10.1038/protex.2016.072
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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 present a method that employs subtiligase to efficiently ligate Cys-free peptides to protein thioesters.
The ability to site-specifically install post-translational modifications, biophysical probes, unnatural amino acids, isotopic labels, and drug-like small molecules into proteins of any size offers enormous potential for both fundamental and applied biomedical research. Semisynthesis using expressed protein ligation (EPL) has been exploited frequently in the construction of proteins containing diverse chemical modifications. In standard EPL, a recombinant protein fragment is fused to an intein, which is then reacted with a thiol to generate the isolated recombinant protein C-terminal thioester. An N-Cys containing synthetic peptide is then added to the protein thioester which undergoes transthioesterifcation followed by rearrangement to a conventional amide bond. While powerful, the scope of EPL is narrowed by the requirement of a Cys at the ligation junction.
Subtiligase is an engineered peptide ligase derived from the protease subtilisin that contains mutations (S221C, P225A) which alter the mechanism to favor aminolysis over peptidase activity, facilitating the ligation of a peptide containing a C-terminal ester or thioester to a peptide containing an α-amine.
We describe here a general method for generating a protein thioester from a protein-intein fusion, and using subtiligase to ligate it to a synthetic peptide.
E. coli DH5α for cloning
E. coli BL21 for expression
SF21 and High Five insect cells for expression
Lysis buffer: 250 mM NaCl, 50 mM HEPES, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, pH = 7.5
Wash buffer: 250 mM NaCl, 25 mM HEPES, 0.1% Triton X-100, pH = 7.5
Cleavage buffer: 250 mM NaCl, 50 mM HEPES, 1 mM EDTA, pH = 7.5
Storage buffer: 150 mM NaCl, 50 mM MES, pH = 6-6.5. The thioester is more stable at a lower pH, but pH up to 6.5 is fine if the protein of interest cannot tolerate a lower pH.
Reaction buffer: 10 mM CaCl2, 100 mM BICINE, pH = 8.0
Chitin Resin
Sodium Mercaptoethane sulfonate (MESNA)
Subtiligase (100 μM stock)
Equipment for protein expression culture
Equipment for cell lysis, e.g. French press cell for E. coli or homogenizer for insect cells.
Floor and table centrifuges
Empty Biorad gravity flow column
Heat block capable of maintaining 25°C
SDS-PAGE system
Creating protein thioester: 24 hours (4 - 6 hours + overnight incubation)
Ligation: 2 - 4 hours
If ligation efficiency is low: reposition the ligation site so that hydrophobic residues are at the 1st and 4th positions upstream of the ligation site. Avoid acidic residues in these positions, or at the C-terminus of the peptide.
Use greater concentrations of peptide and/or subtiligase.
For peptide/proteins with acidic residues at the ligation junction use E156Q,G166K or Y217K variants of subtiligase.
For peptides containing phosphates, use a high-calcium buffer (up to 100 mM).
In all cases, longer reaction times may be necessary.
After analyzing the reaction by SDS-PAGE, there should be three main bands in the reaction lanes: subtiligase (around 27 kDa), the ligation product, and below it, unreacted protein thioester hydrolyzed by subtilgase. See "Figure 2":http://www.nature.com/protocolexchange/system/uploads/4737/original/EnzymeResult.jpg?1472494297 for an example ligation between PTEN and a 25-mer peptide.
This work was supported by the NIH and the FAMRI foundation. We thank Y. Li for assisting with insect cell culture. We thank the other members of the Cole lab for valuable discussions and advice.
No conflicting financial interests
