Mutated PGLB oligosaccharyltransferase enzymes
The present disclosure provides mutated PglB oligosaccharyltransferase enzymes, polynucleotides that encode the mutated PglB oligosaccharyltransferase enzymes, host cells capable of expressing the engineered PglB oligosaccharyltransferase enzymes, N-glycosylated proteins that are made using the engineered PglB oligosaccharyltransferase enzymes, and methods of using the engineered PglB oligosaccharyltransferase enzymes.
1 . A PglB oligosaccharyltransferase (OST) polypeptide comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 comprising:
(a) at least one substituted residue at an amino acid selected from the group consisting of X477, X273, X65, X69, X72, X78, X101, X125, X147, X151, X176, X195, X199, X202, X204, X205, X206, X223, X260, X272, X275, X279, X281, X282, X284, X289, X293, X303, X305, X306, X310, X315, X320, X358, X360, X362, X371, X382, X397, X402, X462, X466, X489, X520, X523, X570, and X605, and wherein the substituted residue is different from the amino acid at the corresponding position in SEQ ID NO: 2; and
(b) amino acid 273 of SEQ ID NO: 2 is mutated to a residue selected from the group consisting of L, D, E, K, and R.
2 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 477 of SEQ ID NO: 2 is mutated to a residue selected from the group consisting of A and S.
3 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 195 of SEQ ID NO: 2 is mutated to a residue selected from the group consisting of R, K, H, and C.
4 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 305 of SEQ ID NO: 2 is mutated to a residue selected from the group consisting of D and R.
5 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 273 of SEQ ID NO: 2 is R.
6 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 477 of SEQ ID NO: 2 is S.
7 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 195 of SEQ ID NO: 2 is R.
8 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 305 of SEQ ID NO: 2 is R.
9 . The PglB OST polypeptide of claim 1 , wherein the residue corresponding to amino acid 195 of SEQ ID NO: 2 is R, the residue corresponding to amino acid 273 of SEQ ID NO: 2 is R, the residue corresponding to amino acid 305 of SEQ ID NO: 2 is R, and the residue corresponding to amino acid 477 is S.
10 . The PglB OST polypeptide of claim 1 , wherein the amino acid sequence is at least 95% identical to SEQ ID NO: 2.
11 . A polynucleotide encoding the PglB OST polypeptide of claim 1 .
12 . A host cell comprising the PglB OST polypeptide of claim 1 or a polynucleotide encoding the PglB OST polypeptide.
13 . The host cell of claim 12 , wherein the host cell is a prokaryotic host cell.
14 . The host cell of claim 12 , wherein the host cell is E. coli.
15 . The host cell of claim 12 , wherein the polynucleotide encoding the PglB OST polypeptide is integrated into the host cell genome or is expressed from a plasmid.