Expression of soluble, active eukaryotic glycosyltransferases in prokaryotic organisms
View Patent ↗The present invention provides enhanced methods of producing soluble, active eukaryotic glycosyltransferases in prokaryotic microorganisms that have an oxidizing environment.
1. A method of producing a soluble truncated active eukaryotic glycosyltransferase in a prokaryotic microorganism, which method comprising the steps of
(a) providing a prokaryotic microorganism that has an oxidizing intracellular environment,
(b) expressing a nucleic acid that encodes a truncated eukaryotic glycosyltransferase in the prokaryotic microorganism, wherein the truncated eukaryotic glycosyltransferase is a truncated human GalNacT2 of SEQ ID NO: 33 having a truncation selected from the group consisting of Δ51 Δ445, or one or two truncations selected from the group consisting of Δ40, Δ53, Δ73, Δ94, and Δ445, and
(c) growing the microorganism under conditions that allow expression of the soluble truncated active eukaryotic glycosyltransferase within a cellular compartment of the prokaryotic microorganism.
2. The method of claim 1 , wherein the truncated human GalNacT2 is selected from the group consisting of, human GalNAcT2 (Δ40), human GalNAcT2(Δ73), human GalNAcT2(Δ94), human GalNAcT2 (Δ51Δ445), human GalNAcT2(Δ53), and human GalNAcT2(Δ53Δ445).
3. A method of producing a soluble active eukaryotic glycosyltransferase fusion protein in a prokaryotic microorganism, which method comprises:
(a) providing a prokaryotic microorganism that has an oxidizing intracellular environment,
(b) expressing a nucleic acid that encodes a soluble active eukaryotic glycosyltransferase fusion protein which comprises a maltose binding protein (MBP) and a truncated eukaryotic glycosyltransferase, wherein the truncated eukaryotic glycosyltransferase is a truncated human GalNAcT2 of SEQ ID NO: 33 having a truncation selected from the group consisting of Δ51Δ445, or one or two truncations selected from the group consisting of Δ40, Δ53, Δ73, Δ94, and Δ445, in the prokaryotic microorganism, and
(c) growing the microorganism under conditions that allow expression of the soluble active eukaryotic glycosyltransferase fusion protein within a cellular compartment of the prokaryotic microorganism.
4. The method of claim 3 , wherein the soluble active eukaryotic glycosyltransferase fusion protein is an 0 -linked glycan glycosyltransferase fusion protein having human MBP-GalNAc-T2 (Δ51, Δ445).
5. The method of claim 4 , wherein the soluble active human MBP-GalNAc-T2 (Δ51, Δ445) has an enzyme activity of about 5 U/L.