Protein engineering enables Serratia marcescens nuclease A to hydrolyze nucleic acids under high-salt conditions

For the purpose of removing nucleotide impurities, Serratia marcescens Nuclease A (SmNucA) is widespread used in biopharmaceutical manufacturing, such as production of adeno-associated viral vector, one of the leading gene delivery platforms that features low immunogenicity. However, such utilization of wild-type SmNucA is limited in saline environment. Depending on downstream process development, the ionic strength can be as high as 500 mM, at which the potency of wild-type SmNucA plunges. We herein design an SmNucA variant with four Lys mutations, namely HighSalt NucA, to improve nucleic acids binding under high-salt conditions. K_m determination and molecular dynamics (MD) simulation implies a new catalytical mechanism adopted by HighSalt NucA and another mutant that harbors four Arg substitutions at the same sites. We thereby conclude that basic-residue mutations on the SmNucA surface stabilize the local conformation in close proximity to the substrate-binding cleft at saline concentration of 500 mM. In addition to Lys and Arg mutations, saturation mutagenesis further indicated that certain hydrophobic residues (Ala, Val, Trp, Tyr) and polar residues (Ser, Thr, Asn, Gln) also render SmNucA salt-tolerant, albeit to differing extent. In contrast to activity loss of the wild-type with 400-500 mM NaCl, HighSalt NucA maintains broad substrate specificity even under these extreme conditions, which expands its application prospect in the removal of nucleic acid impurities during process development.