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Publication Number
US-12509675-B2
Publication Date
2025-12-30
Expiration Date
Abstract
The invention relates to aminoacyl-tRNA synthetases that aminoacylate tRNA with 2-Aminoisobutyric acid (Aib), thus enabling the incorporation of the Aib into a growing polypeptide chain during translation, e.g. in eubacterial host cells such as E. coli. For example, but not limited to, the invention relates to new aminoacyl-tRNA synthetases and the uses hereof, as well as a method for producing polypeptides that contain one or more Aib.
Core Innovation
The invention relates to a 2-Aminoisobutyric acid-tRNA synthetase (AibRS) comprising an amino acid sequence of SEQ ID NO: 7 or a variant thereof, wherein the variant retains 215Gly and is at least 90% identical to SEQ ID NO: 7. The AibRS aminoacylates tRNA with 2-aminoisobutyric acid (Aib), thereby enabling translation of Aib into polypeptides. A suppressor tRNA is described, including an anticodon complementary to stop codons such as TAG (amber).
The disclosed system further specifies a defined set of AibRS variants that preserve Aib aminoacylation and retain 215Gly. Variant structure is constrained by formula-based frameworks and residue-combination restrictions, including allowed amino-acid substitutions at positions 192, 193, 213, 216, 217, 249, 360, and 459, as well as selection from enumerated SEQ ID NO: 10 to SEQ ID NO: 30.
The disclosed content also describes testing AibRS mutants of the phAlaRS class II N-terminal aa1-495 domain for Aib incorporation into model polypeptides. Model polypeptides include IL21-H(Aib)-GLP-1 and MS-(Aib)-hsLeptin, and incorporation is confirmed by LC-MS and SDS-PAGE. The system is further applied to produce and derivatize GLP-1 semaglutide (SEQ ID NO: 32) and related compounds via derivatization with a specified substituent (Chem. 3).
Claims Coverage
The consolidated claim coverage centers on an AibRS that aminoacylates tRNA with Aib using an SEQ ID NO: 7-based sequence or variant set. Across the independent and dependent claims, the inventive features focus on the SEQ ID NO: 7 sequence constraints, retention of 215Gly, the at-least-90% identity requirement, residue-position restrictions, selection from enumerated SEQ ID numbers, and suppressor tRNA as the tRNA substrate.
AibRS sequence-constrained Aib aminoacylation
A 2-Aminoisobutyric acid-tRNA synthetase (AibRS) comprising an amino acid sequence of SEQ ID NO: 7 or a variant thereof, having the function of aminoacylating tRNA with 2-aminoisobutyric acid (Aib), wherein the variant retains 215Gly and is at least 90% identical to SEQ ID NO: 7.
Position-specific residue substitutions in the SEQ ID NO: 7 variant
The AibRS variant is defined by specific amino-acid substitutions at positions 192, 193, 213, 216, 217, 249, 360, and 459, each chosen from the stated sets.
Enumerated SEQ ID NO: 7 variant residue combinations
The SEQ ID NO: 7 variant contains specific amino-acid substitutions at positions 192, 215, and related sites including 360 and 459, with multiple allowed combinations enumerated.
Finite set of permitted SEQ ID numbers for AibRS
The variant of SEQ ID NO: 7 is selected from a list consisting of SEQ ID NO: 10, SEQ ID NO: 11, 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, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.
Suppressor tRNA as the tRNA substrate
The tRNA involved is a suppressor tRNA.
Overall, the claim set is anchored by an AibRS defined by SEQ ID NO: 7 or a variant that aminoacylates tRNA with Aib while retaining 215Gly and meeting at least 90% identity. Dependent claims narrow the variant space using position-specific substitution rules, enumerated residue combinations, an explicit finite list of permitted SEQ ID numbers, and suppressor tRNA.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Production and derivatization of GLP-1 semaglutide (SEQ ID NO: 32) and related compounds using derivatization with a specified substituent (Chem. 3).
Expression of Aib-containing model GLP-1 constructs with characterization via LC-MS incorporation ratio analysis (Aib vs Ala-containing peaks).
[procedural detail omitted for safety]
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