Available online 1 August 2022
Highlights•Bacterial selection system used to increase catalytic turnover of a protease for fusion protein processing
•Four point-mutations were identified for the activity increase
•The modified protease retains the high manufacturability of the original enzyme
•Tag removal can be performed regardless of target protein, creating a native N-terminus
AbstractFusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generated the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.
AbbreviationsATCaseaspartate carbamoyltransferase
BCAAbranched chain amino acids
CASPONcaspase-based fusion
cpCasp2circularly permuted caspase-2
DSCdifferential scanning calorimetry
E2ubiquitin-conjugating enzyme E2 L3
FGF2fibroblast growth factor 2
FRETFörster resonance energy transfer
GCSFgranulocyte colony stimulating factor
IMACimmobilized metal affinity chromatography
IPTGisopropyl β-D-1-thiogalactopyranoside
PROFICSprotease optimization via fusion-inhibited carbamoyltransferase-based selection
KeywordsLaboratory evolution
affinity tag removal
protease
E. coli
recombinant protein
platform process
microbial expression system
© 2022 The Authors. Published by Elsevier B.V.
留言 (0)