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Publication Number
US-11572552-B2
Publication Date
2023-02-07
Expiration Date
Abstract
Reported herein is a sortase comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 and that comprises the mutations D101S and K137S.
Core Innovation
The invention relates to a sortase variant comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 11 and carrying substitutions of residues D101 and K137 to D101S and K137S, respectively, with the residue positions based on the amino acid sequence of SEQ ID NO: 11. By introducing these substitutions into a shortened Sortase A sequence, the invention obtains a sortase with improved enzymatic activity compared to a sortase comprising the amino acid sequence of SEQ ID NO: 11.
The disclosed core includes additional mutational embodiments extending beyond D101S and K137S, with one or more additional mutation sites specified relative to SEQ ID NO: 11. Enumerated additional mutation embodiments include substitutions such as E106G, N107W, F144L, G167E, and variants combining these with D160S and K196S or with D101S and K137S, together with other additional mutation site sets described relative to SEQ ID NO: 11.
The invention further includes embodiments based on mutating full-length S. aureus Sortase A (SEQ ID NO: 01) at D160S and K196S. The document reports improved catalytic performance versus wild-type, with activity, stability, and apparent Km behavior discussed in relation to recognition and acceptor substrates in the context of sortase-mediated transpeptidation using LPXTG and glycine-based acceptors.
Claims Coverage
The document provides one independent claim directed to improving enzymatic activity of a sortase by providing a variant at least 90% identical to SEQ ID NO: 11 and mutating residues D101 and K137 to D101S and K137S, respectively; dependent claims further define additional mutation sites, constrained mutation sets, and enumerated improved sortase amino acid sequences.
Improved enzymatic activity sortase variant at 90% identity with SEQ ID NO: 11
A method for improving enzymatic activity of a sortase comprising providing a sortase comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 11 and mutating residues D101 and K137 to D101S and K137S, respectively, wherein said amino acid positions are based on the amino acid sequence of SEQ ID NO: 11, thereby obtaining a sortase with improved enzymatic activity as compared to a sortase comprising the amino acid sequence of SEQ ID NO: 11.
Improved enzymatic activity defined by transpeptidation performance attributes
The method further defines an engineered sortase with improved enzymatic activity including increased transpeptidation speed, increased transpeptidation turnover rate, altered substrate affinity, or increased enzymatic activity in a sortase-mediated coupling reaction.
Additional specified mutation sites relative to SEQ ID NO: 11
The method further defines using a sortase with improved enzymatic activity that includes specific mutations at amino acid positions A2, E47, N48, F85, and G108 as defined relative to SEQ ID NO: 11.
Constrained multi-mutation sets for improved enzymatic activity
The method further specifies that a sortase variant with improved enzymatic activity is obtained using one or more specified sets of mutations chosen from the listed groups.
Enumerated improved sortase amino acid sequence embodiments
The method is performed using a sortase with improved enzymatic activity that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.
Overall, the claim coverage centers on engineering sortase variants that are at least 90% identical to SEQ ID NO: 11 and include D101S and K137S substitutions to achieve improved enzymatic activity, with dependent claims further requiring defined additional mutation sites, specific allowable mutation sets, and selection from enumerated SEQ ID NO sequences representing the improved variants.
Stated Advantages
Improved enzymatic activity compared to a sortase comprising the amino acid sequence of SEQ ID NO: 11.
Increased transpeptidation speed.
Increased transpeptidation turnover rate.
Altered substrate affinity.
Increased enzymatic activity in a sortase-mediated coupling reaction.
Documented Applications
Sortase-mediated coupling or conjugation reaction using an LPXTG motif and glycine-based acceptors in the context of transpeptidation or transamidation; the document discusses recognition and acceptor substrates including apparent Km behavior for LPXTG and GG.
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