CASPON platform technology: Ultrafast circularly permuted caspase-2 cleaves tagged fusion proteins before all 20 natural amino acids at the N-terminus

Elsevier

Available online 1 August 2022

New BiotechnologyHighlights•

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

Abstract

Fusion 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.

AbbreviationsATCase

aspartate carbamoyltransferase

BCAA

branched chain amino acids

CASPON

caspase-based fusion

cpCasp2

circularly permuted caspase-2

DSC

differential scanning calorimetry

E2

ubiquitin-conjugating enzyme E2 L3

FGF2

fibroblast growth factor 2

FRET

Förster resonance energy transfer

GCSF

granulocyte colony stimulating factor

IMAC

immobilized metal affinity chromatography

IPTG

isopropyl β-D-1-thiogalactopyranoside

PROFICS

protease optimization via fusion-inhibited carbamoyltransferase-based selection

Keywords

Laboratory evolution

affinity tag removal

protease

E. coli

recombinant protein

platform process

microbial expression system

© 2022 The Authors. Published by Elsevier B.V.

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