On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

Abstract

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed ‘OaAEP1b‐C247A‐∆351’ could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

“…To obtain soluble protein, intact AEP zymogens, rather than the catalytic core domain itself, are typically overexpressed in bacteria or other host cells, because the core domain alone is less stable and thus prone to forming inclusion bodies. To date, only one paper has reported the successful renaturation of the catalytic core domain of OaAEP1 from inclusion bodies [51]; however, whether this approach can be applied to other AEP‐type peptide ligases is unknown. We have tested this approach using the core domain of butelase‐1, but no ligase activity was detected after in vitro renaturation using the reported procedure.…”