Hoffmann-la Roche Inc.

Martin Schatte

Mara Boenitz-Dulat

Peter Kratzsch

Erhard Kopetzki

Frank Bergmann

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.

Sortase

Herein is reported a polypeptide comprising the amino acid sequence of SEQ ID NO: 38 as solederived polypeptide and its use in conjugating polypeptides.

Soluble Sortase A

Herein is reported a method for the enzymatic production of a polypeptide comprising the step of incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue) or LPXTA (SEQ ID NO: 41, wherein X can be any amino acid residue), ii) a second polypeptide that has i) a glycinyl, an alaninyl, or a cysteinyl compound at its N-terminus, or ii) an oligoglycine, or oligoalanine, or a cysteine amino acid residue followed by one to three glycine or alanine amino acid residues at its N-terminus, or iii) a lysine amino acid residue within its 5 N-terminal amino acid residues, and iii) a third polypeptide with sortase A activity, in a deep eutectic solvent and thereby producing a polypeptide.

Transamidation reaction in deep eutectic solvents

Herein is reported a method for the detection of a sortase in a sample, comprising the following steps:

Activity assay for bond forming enzymes

Herein is reported a method for the enzymatic production of a thioester comprising incubating i) a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 01, wherein X can be any amino acid residue), ii) a second polypeptide that has at its N-terminus a cysteine amino acid residue or is a cysteinyl compound, and iii) a third polypeptide with sortase A activity.

Production of thioesters using sortase

Herein is reported a method for producing an enzymatic conjugation product of two polypeptides comprising incubating of a first polypeptide comprising the amino acid sequence LPXTG (SEQ ID NO: 20, wherein X can be any amino acid residue), a second polypeptide has an oligo-alanine A(m=2 (SEQ ID NO: 26), or 3 (SEQ ID NO: 27), or 4 (SEQ ID NO: 28), or 5 (SEQ ID NO: 29)) amino acid sequence at its N-terminus, a third polypeptide with sortase activity which is derived fromSortase A, and recovering the conjugate from the reaction mixture and thereby producing the enzymatic conjugation product of two polypeptides.

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Karlsbad, Germany

Martin Schatte