Caspase-Based Fusion Protein Technology: Substrate Cleavability Described by Computational Modeling and Simulation.

The Caspase-based fusion protein technology (CASPON) allows for universal cleavage of fusion tags from proteins of interest to reconstitute the native N-terminus. While the CASPON enzyme has been optimized to be promiscuous against a diversity of N-terminal peptides, the cleavage efficacy for larger proteins can be surprisingly low. We develop an efficient means to rationalize and predict the cleavage efficiency based on a structural representation of the intrinsically disordered N-terminal peptides and their putative interactions with the CASPON enzyme. The number of favorably interacting N-terminal conformations shows a very good agreement with the experimentally observed cleavage efficiency, in agreement with a conformational selection model. The method relies on computationally cheap molecular dynamics simulations to efficiently generate a diverse collection of N-terminal conformations, followed by a simple fitting procedure into the CASPON enzyme. It can be readily used to assess the CASPON cleavability a priori.

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

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 generates 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.

Molecular Dynamics and In Vitro Quantification of Safrole DNA Adducts Reveal DNA Adduct Persistence Due to Limited DNA Distortion Resulting in Inefficient Repair.

The formation and repair of N2-(trans-isosafrol-3'-yl)-2'-deoxyguanosine (S-3'-N2-dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'-N2-dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'-N2-dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'-N2-dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

Cellular levels and molecular dynamics simulations of estragole DNA adducts point at inefficient repair resulting from limited distortion of the double-stranded DNA helix.

Estragole, naturally occurring in a variety of herbs and spices, can form DNA adducts after bioactivation. Estragole DNA adduct formation and repair was studied in in vitro liver cell models, and a molecular dynamics simulation was used to investigate the conformation dependent (in)efficiency of N2-(trans-isoestragol-3'-yl)-2'-deoxyguanosine (E-3'-N2-dG) DNA adduct repair. HepG2, HepaRG cells, primary rat hepatocytes and CHO cells (including CHO wild-type and three NER-deficient mutants) were exposed to 50 µM estragole or 1'-hydroxyestragole and DNA adduct formation was quantified by LC-MS immediately following exposure and after a period of repair. Results obtained from CHO cell lines indicated that NER plays a role in repair of E-3'-N2-dG adducts, however, with limited efficiency since in the CHO wt cells 80% DNA adducts remained upon 24 h repair. Inefficiency of DNA repair was also found in HepaRG cells and primary rat hepatocytes. Changes in DNA structure resulting from E-3'-N2-dG adduct formation were investigated by molecular dynamics simulations. Results from molecular dynamics simulations revealed that conformational changes in double-stranded DNA by E-3'-N2-dG adduct formation are small, providing a possible explanation for the restrained repair, which may require larger distortions in the DNA structure. NER-mediated enzymatic repair of E-3'-N2-dG DNA adducts upon exposure to estragole will be limited, providing opportunities for accumulation of damage upon repeated daily exposure. The inability of this enzymatic repair is likely due to a limited distortion of the DNA double-stranded helix resulting in inefficient activation of nucleotide excision repair.