CELL-FREE ENZYMATIC METHOD FOR PREPARATION OF N-GLYCANS

Abstract

The present invention relates to a cell-free enzyme-catalyzed process for producing glycoproteins of general formula (I) from a lipid-linked oligosaccharide and a peptide. Further, said process includes the construction of the lipid-linked oligosaccharide from a mannose trisaccharide containing core structure. Particularly, the lipid-linked oligosaccharide is a high mannose-, complex-, or hybrid-type N-glycan.

Claims

1. An in vitro method for producing a glycoprotein of general formula (I) ##STR00221## wherein C represents a carbohydrate of the following structure ##STR00222## o is an integer representing the number of carbohydrates C which are bound to a peptide P, P represents a peptide of at least 20 amino acids comprising at least o-times a consensus sequence of N-X-S/T, wherein X represents any amino acid except proline, and NH represents an asparagine -amido group of the consensus sequence; F.sup.1 and F.sup.2 represent ##STR00223## or H; with the proviso that F.sup.1 and F.sup.2 cannot be simultaneously ##STR00224## G represents ##STR00225## or H; S.sup.1 represents ##STR00226## S.sup.2 represents ##STR00227## S.sup.3 represents ##STR00228## S.sup.4 represents ##STR00229## S.sup.5 represents ##STR00230## M.sup.S1 represents ##STR00231## M.sup.S2 represents ##STR00232## M.sup.S3 represents ##STR00233## M.sup.S4 represents ##STR00234## M.sup.S5 represents ##STR00235## L.sup.S1 represents ##STR00236## L.sup.S2 represents ##STR00237## L.sup.S3 represents ##STR00238## L.sup.S4 represents ##STR00239## L.sup.S5 represents ##STR00240## T.sup.S1, T.sup.S2 T.sup.S3 T.sup.S4 and T.sup.S5 represent independently of each other: ##STR00241## SO.sub.4 or a bond wherein m.sub.S1, m.sub.S2, m.sub.S3, m.sub.S4, m.sub.S5, I.sub.S1, I.sub.S2, I.sub.S3, I.sub.S4, and I.sub.S5 represent independently of each other an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, n.sub.S1, n.sub.S2, n.sub.S3, n.sub.S4 and n.sub.S5 represent an integer selected from 0 and 1, with the proviso that if n.sub.S3=1 then n.sub.S1=1 and m.sub.S2=m.sub.S4=m.sub.S5=0; and if n.sub.S3=0 then i) n.sub.S2=m.sub.S2=m.sub.S3=0, and ii) m.sub.S1>3 when m.sub.S4+m.sub.S5=0; comprising the steps: A) providing a solution comprising a compound of formula (II) ##STR00242## wherein R represents ##STR00243## with a being an integer selected from 2, 3, 4, 5, 6, 7, or 8, B) reacting the compound of formula (II) with the peptide P in the presence of an eukaryotic oligosaccharyltransferase enzyme to produce the compound of formula (I).

2. The method according to claim 1, wherein the consensus sequence of peptide P is N-V-T or N-Y-T.

3. The method according to claim 1, wherein o represents 1.

4. The method according to claim 1 wherein the compound of formula (II) is obtained from a compound for formula (III) ##STR00244## wherein R represents ##STR00245## with a being an integer selected from 2, 3, 4, 5, 6, 7, or 8 by mixing a solution comprising the compound of formula (III) with at least one nucleotide sugar and at least one glycosyltransferase enzyme and reacting a resulting mixture to produce a compound of formula (II) wherein the at least one nucleotide sugar is selected from GDP-mannose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, CMP-NeuAc and CMP-NeuGc, wherein the at least one glycosyltransferase enzyme is selected from N-acetylglucosaminyltransferase, mannosyltransferase, glucosyltransferase, galactosyltransferase, fucosyltransferase and sialyltransferase.

5. The method according to claim 4, wherein the at least one glycosyltransferase enzyme is a transmembrane domain-deleted enzyme.

6. The method according to claim 4, wherein the N-acetylglucosaminyltransferase is an -1,3-mannosyl-glycoprotein 2--N-acetylglucosaminyltransferase and/or an -1,6-mannosyl-glycoprotein 2--N-acetylglucosaminyltransferase and/or a -1,4-mannosyl-glycoprotein 4--N-acetylglucosaminyltransferase and/or an -1,3-mannosyl-glycoprotein 4--N-acetylglucosaminyltransferase, wherein the mannosyltransferase is an -1,2-mannosyltransferase and/or an -1,3-mannosyltransferase and/or an -1,6-mannosyltransferase and/or an -1,3/1,6-mannosyltransferase, wherein the galactosyltransferase is an -1,3-galactosyltransferase and/or a -1,4-galactosyltransferase, wherein the sialyltransferase is an -2,3-sialyltransferase and/or an -2,6-sialyltransferase, wherein the fucosyltransferase is an -1,2-fucosyl-transferase and/or an -1,3-fucosyltransferase and/or an -1,6-fucosyltransferase, and wherein the glucosyltransferase is an -1,2-glucosyltransferase and/or an -1,3-glucosyltransferase.

7. The method according to claim 1, wherein P is a therapeutic protein or wherein P consists of at least 50 amino acids.

8. The method according to claim 1, wherein the oligosaccharyltransferase enzyme is STT3A protein from Trypanosoma brucei.

9. The method according to claim 1, wherein T.sup.S1, T.sup.S2, T.sup.S3, T.sup.S4, and T.sup.S5 represent a bond.

10. The method according to claim 1, wherein I.sub.S1, I.sub.S2, I.sub.S3, I.sub.S4, and I.sub.S5 represent independently of each other an integer selected from 0, 1, 2 and 3.

11. The method according to claim 1, wherein F.sup.1, F.sup.2 and G represent H.

12. The method according to claim 1, wherein the peptide P is aglycosylated.

13. The method according to claim 1, wherein n.sub.S1=0; n.sub.S2=m.sub.S2=0; n.sub.S3=m.sub.S3=0; n.sub.S4=0; n.sub.S5=0; wherein m.sub.S1, m.sub.S4, and m.sub.S5 represent independently of each other an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, with the proviso that m.sub.S1>3, when m.sub.S4+m.sub.S5=0.

14. An in vitro method for producing a compound of general formula (IIIb) ##STR00246## wherein F.sup.1 and F.sup.2 represent ##STR00247## or H; with the proviso that F.sup.1 and F.sup.2 cannot be simultaneously ##STR00248## G represents ##STR00249## or H; Sb.sup.1 represents ##STR00250## Sb.sup.2 represents ##STR00251## or H Sb.sup.3 represents ##STR00252## Sb.sup.4 represents ##STR00253## or H L.sup.Sb1 represents ##STR00254## L.sup.Sb2 represents ##STR00255## L.sup.Sb3 represents ##STR00256## L.sup.Sb4 represents ##STR00257## T.sup.Sb1, T.sup.Sb2, T.sup.Sb3 and T.sup.Sb4 represent independently of each other: ##STR00258## SO.sub.4 or a bond wherein I.sub.S1, I.sub.S2, I.sub.S3, and I.sub.S4 represent independently of each other an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, comprising the steps: Ab) providing a solution comprising a compound of formula (IIb) ##STR00259## wherein R represents ##STR00260## with a being an integer selected from 2, 3, 4, 5, 6, 7, or 8, Bb) mixing the solution with at least one nucleotide sugar and at least one glycosyltransferase enzyme and reacting a resulting mixture to produce the compound of formula (IIIb), wherein the at least one nucleotide sugar is selected from GDP-mannose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, CMP-NeuAc and CMP-NeuGc, and wherein the at least one glycosyltransferase enzyme is selected from N-acetylglucosaminyltransferase, mannosyltransferase, glucosyltransferase, galactosyltransferase, fucosyltransferase and sialyltransferase.

15. An in vitro method for producing a compound of general formula (Ic) ##STR00261## wherein C represents a carbohydrate of the following structure ##STR00262## o is an integer representing the number of carbohydrates C which are bound to a peptide P, P represents a peptide of at least 20 amino acids comprising at least o-times a consensus sequence of N-X-S/T, wherein X represents any amino acid except proline, and NH represents an asparagine -amido group of the consensus sequence wherein F.sup.1 and F.sup.2 represent ##STR00263## or H; with the proviso that F.sup.1 and F.sup.2 cannot be simultaneously ##STR00264## G represents ##STR00265## or H; Sc.sup.1 represents ##STR00266## Sc.sup.2 represents ##STR00267## or H Sc.sup.3 represents ##STR00268## Sc.sup.4 represents ##STR00269## or H L.sup.Sc1 represents ##STR00270## L.sup.Sc2 represents ##STR00271## L.sup.Sc3 represents ##STR00272## L.sup.Sc4 represents ##STR00273## T.sup.Sc1, T.sup.Sc2, T.sup.Sc3, and T.sup.Sc4 represent independently of each other: ##STR00274## wherein I.sub.S1, I.sub.S2, I.sub.S3, and I.sub.S4 represent independently of each other an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, comprising the steps: Ac) providing a solution comprising a compound of formula (IIc) ##STR00275## wherein R represents ##STR00276## with a being an integer selected from 2, 3, 4, 5, 6, 7, or 8, Bc) reacting the compound of formula (IIc) with the peptide P in the presence of a eukaryotic oligosaccharyltransferase enzyme to produce the compound of formula (IVc) ##STR00277## wherein Cc represents a carbohydrate of the following structure ##STR00278## Cc) mixing the compound of formula (IVc) with at least one nucleotide sugar and at least one glycosyltransferase enzyme and reacting a resulting mixture to produce the compound of formula (Ic), wherein the at least one nucleotide sugar is selected from GDP-mannose, UDP-galactose, UDP-GlcNAc, UDP-GalNAc, GDP-fucose, CMP-NeuAc and CMP-NeuGc, and wherein the at least one glycosyltransferase enzyme is selected from N-acetylglucosaminyltransferase, mannosyltransferase, glucosyltransferase, galactosyltransferase, fucosyltransferase and sialyltransferase.

Description

DESCRIPTION OF THE FIGURES

[1578] FIG. 1: shows the general structure of the three types of N-glycans: high mannose, complex-type and hybrid-type.

[1579] FIG. 2: shows exemplarily the structure of glycans prepared with the inventive methods

[1580] FIG. 3: shows a xCGE-LIF electropherogram of LL-GlcNAc.sub.2Man.sub.3 synthesis. The reaction was conducted in triplicates (Man.sub.3-1/2/3) for 8 h in a reaction batch initially, containing 50 mM MOPS (pH 6.8), 0.1% IGEPAL, 10 mM MgCl.sub.2 (buffer B), 1 mM DTT, 0.1 mM phytanyl-PP-chitobiose, 2 mM GDP-mannose, 0.1 mg/mL purified ALG1TM and 35% (v/v) ALG2 yeast membrane fraction. Samples were prepared for CGE-LIF measurements by mild acidic hydrolysis, APTS labeling followed by HILIC purification. Internal LIZ and 2.sup.nd NormMix standards were used for analysis using glyXtool.sup.CE. N-glycans are depicted according to the SNFG-nomenclature (Glycobiology 2015, 25, 1323-1324).

[1581] FIG. 4: shows a xCGE-LIF electropherogram showing the synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc[3] with MGAT1 (red) and LL-GlcNAc.sub.2Man.sub.3GlcNAcGal[3] with MGAT1+4GalT1 (blue). Internal LIZ and 2nd NormMix standards were used for analysis using glyXtoolCE. N-glycans are depicted according to the SNFG-nomenclature (Glycobiology 2015, 25, 1323-1324).

[1582] FIG. 5: shows a xCGE-LIF electropherogram showing the synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 with MGAT1+2 (red) and LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 with MGAT1+2 and 4GalT1. Internal LIZ and 2nd NormMix standards were used for analysis using glyXtoolCE. N-glycans are depicted according to the SNFG-nomenclature (Glycobiology 2015, 25, 1323-1324).

[1583] FIG. 6: shows a xCGE-LIF electropherogram showing the one-pot synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 at 0 h (black), 4 h (red), 8 h (blue), 24 h (orange) and 36 h (violet). Signal intensity was plotted against the migration time. Internal LIZ and 2nd NormMix standards were used for analysis using glyXtoolCE. N-glycans are depicted according to the SNFG-nomenclature (Glycobiology 2015, 25, 1323-1324).

[1584] FIG. 7: shows the relative quantification of in vitro N-glycosylated synthetic TAMRA peptides for four different enzyme reactions (A: MGAT1, B: MGAT1+B4GALT, C: MGAT1+2, D: MGAT1+2+B4GALT). Extracted ion chromatograms (EIC MS.sup.1) of the precursor ions of TAMRA+GSDANYTYTQ (SEQ ID NO 1)+N-glycan are depicted for each enzymatic reaction and show the retention and relative abundance of the different N-glycoforms. For each EIC the peak intensity in arbitrary units (e.g. 9.2510.sup.5) is given for the respective glycopeptide precursor ions. Only EIC-MS.sup.1 precursor ions with MS.sup.2 spectra are selected.

[1585] FIG. 8: shows Tris-Tricine PAGE of high-mannose LL-GlcNAc2Man3/Man5, with 21-mer peptide. The negative control included heat inactivated OST as a control of aglycosylated peptide and to verify OST activity. The assay was conducted in triplicates.

[1586] FIG. 9: shows an electropherogram of reaction products of N-glycosylation of a 100 aa HA1 protein) after PNGase F digestion and CGE-LIF analysis. Man.sub.3 (178 MTU) and Man.sub.5 (248 MTU) glycans were detected in all three triplicates, but not in the negative control. The peak at 212 MTU can be assigned to a side product which only occurred in samples from SDS gels.

[1587] FIG. 10: shows an electropherogram of reaction products of N-GlcNAcylation of 100 aa HA1 protein by MGAT1 & MGAT2 after PNGase F digestion and CGE-LIF analysis. A1G0 (GlcNAc.sub.2Man.sub.3GlcNAc) (218 MTU) and A2G0 (GlcNAc.sub.2Man.sub.3GlcNAc.sub.2) (252 MTU) glycans were detected in all three triplicates, but not in the negative control.

[1588] FIG. 11: shows an electropherogram of galactosylation reaction products (in-vitro glycoengineering 100 aa HA1 protein (HA1-A1G0 & HA1-A2G0)) after PNGase F digestion and CGE-LIF analysis. A1G1 (GlcNAc.sub.2Man.sub.3GlcNAcGal-peptide) (262 MTU) and A2G2 (GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2-peptide) (331 MTU) glycans were detected in all three triplicates, but not in the negative control.

[1589] FIG. 12: shows an electropherogram of Neu5Acylation reaction products (in-vitro glycoengineering 100 aa HA1 protein (HA1-A1G1 & HA1-A2G2)) after PNGase F digestion and CGE-LIF analysis. A2G2S2 (GlcNAc.sub.2Man.sub.3GlcNAcGalNeu5Ac) (167 MTU) and A2G2S1 (GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2Neu5Ac.sub.2) (229 MTU) glycans were detected, but not in the negative control.

[1590] FIG. 13: shows an electropherogram of the reaction products of an in vitro glycoengineering of lipid-linked Man.sub.3 by an enzymatic cascade using MGAT1, MGAT2 & MGAT5 followed by the addition of 4GalT1 after CGE-LIF analysis.

[1591] FIG. 14: shows an electropherogram of reaction products (in-vitro glycoengineering of HA1 peptides (Man.sub.3 glycan) using MGAT1) after xCGE-LIF analysis.

[1592] FIG. 15: shows an electropherogram of reaction products (in-vitro glycoengineering of HA1 peptides (A1G0 glycan) using MGAT2) after xCGE-LIF analysis.

[1593] FIG. 16: shows an electropherogram of reaction products (in-vitro glycoengineering of HA1 peptides (A2G0 glycan) using b4GalT1) after xCGE-LIF analysis.

[1594] FIG. 17: shows an electropherogram of reaction products (in-vitro glycoengineering of HA1 peptides (A2G2 glycan) using ST6Gal1) after xCGE-LIF analysis.

[1595] FIG. 18: shows an electropherogram of reaction products (in-vitro glycoengineering of lipid-linked Man.sub.3 (black) by an enzymatic cascade using MGAT1, MGAT2 & MGAT5 (gray)) after CGE-LIF analysis.

[1596] FIG. 19: shows an electropherogram of reaction products (in-vitro glycoengineering of lipid-linked Man.sub.3 modified by MGAT1, MGAT2 & MGAT5 (black) followed by the addition of b4GalT1 (gray)) after CGE-LIF analysis.

[1597] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples, which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments, which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[1598] Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

Abbreviations and Acronyms

[1599] % (v/v) volume percent [1600] ACN Acetonitrile [1601] ALG Asparagine-linked glycosylation [1602] ALG1 -1,4-mannosyltransferase [1603] ALG1TM Transmembrane deleted -1,4-mannosyltransferase [1604] ALG2 -1,3/1,6-mannosyltransferase [1605] APTS (3-Aminopropyl)triethoxysilane [1606] 4GalT1TM transmembrane domaine deleted -1,4-galactosyltransferase 1 [1607] CGE-LIF capillary gel electrophoresis with laser-induced fluorescence detection [1608] CHS 3-Hydroxy-5-cholestene 3-hemisuccinate [1609] CMP Cytidine monophosphate [1610] CMP-Neu5Ac Cytidine monophosphate-N-acetylneuraminic acid [1611] CMP-NeuAc Cytidine monophosphate-N-acetylneuraminic acid [1612] CMP-NeuGc Cytidine monophosphate-N-glycolyl neuraminic acid [1613] DDM n-Dodecyl--D-maltopyranoside [1614] DTT Dithiothreitol [1615] E. coli Escherichia coli [1616] ER Endoplasmic reticulum [1617] Gal Galactose [1618] GDP Guanosine diphosphate [1619] GlcNAc N-Acetylglucosamine [1620] GT Glycosyltransferase [1621] HiDi Hi-Di Formamide [1622] HILIC Hydrophilic interaction chromatography [1623] IMAC Immobilized metal affinity chromatography [1624] IPTG Isopropyl -D-1-thiogalactopyranoside [1625] LC-MS Liquid Chromatography coupled mass spectrometry [1626] LLO Lipid-linked oligosaccharide [1627] Man Mannose [1628] MGAT1TM transmembrane domaine deleted -1,3-mannosyl-glycoprotein 2--N-acetylglucosaminyltransferase [1629] MGAT2TM transmembrane domaine deleted -1,6-mannosyl-glycoprotein 2--N-acetylglucosaminyltransferase [1630] MgCl.sub.2 Magnesium chloride [1631] MnCl.sub.2 Manganese chloride [1632] MWCO Molecular weight cut off [1633] OD600 Optical density at 600 nm [1634] OST Oligosaccharyltransferase [1635] PTM Post-translational modification [1636] rpm Rounds per minute [1637] S. cerevisiae Saccharomyces cerevisiae [1638] ssOST Single-subunit oligosaccharyltransferase [1639] TAMRA 5-Carboxytetramethylrhodamine [1640] TFA Trifluoroacetic acid [1641] UDP-Gal Uridindiphosphate-galactose [1642] UDP-GlcNAc Uridindiphosphate-N-Acetylglucosamine [1643] YFP Yellow fluorescent protein

Chemicals & Reagents

TABLE-US-00001 Chemical Source of origin 2nd NormMix glyXera GmbH 4GalT1TM in pET-28a(+) BioCat Acetonitril VWR Chemicals Agar-Agar powder Carl Roth GmbH APTS Sigma-Aldrich Baculovirus Insect Cell Medium Novagen r BioGel P-10 Media Bio-Rad BL21(DE3) Competent E. coli New England Biolabs GmbH CHS Sigma-Aldrich DDM Thermo Fisher Scientific DTT Sigma-Aldrich Ethanol Carl Roth GmbH GDP-Man Sigma-Aldrich GeneScanTM 500 LIZ Size Standard Applied Biosystems Glycerol Carl Roth GmbH HCl VWR Chemicals HEPES Carl Roth GmbH Hi-Di Formamide Thermo Fisher Scientific IGEPAL CA-630 Sigma-Aldrich Imidazol Sigma-Aldrich IPTG Sigma-Aldrich Isopropanol Sigma-Aldrich K.sub.2HPO.sub.4 Carl Roth GmbH Kanamycin sulfate Carl Roth GmbH KH.sub.2PO.sub.4 Carl Roth GmbH Lemo21(DE3) Competent E. coli New England Biolabs GmbH L-rhamnose New England Biolabs GmbH Magnesium chloride Sigma-Aldrich Manganese chloride Merck Methanol Fisher Scientific MGAT1TM in pET-28a(+) BioCat MGAT2TM in pET-28b(+) BioCat MOPS Carl Roth GmbH NaOH Carl Roth GmbH Phytanyl-PP-Chitobiose Chiroblock Pierce BCA Protein Assay Kit Thermo Fisher Scientific Reducing Agent glyXera GmbH S. cerevisiae ORF ALG2 (YGL065C) Horizon Shuffle T7 Express LysY Competent New England Biolabs GmbH E. coli Sodium chloride Carl Roth GmbH TEA AppliChem Panreac TFA Sigma-Aldrich Tryptone Carl Roth GmbH UDP-Gal Sigma-Aldrich UDP-GlcNac Sigma-Aldrich Yeast extract Carl Roth GmbH

Methods

1. Gene expression and enzyme purification
1.1 Gene expression of ALG1TM, MGAT1TM, 4GalT1TM and MGAT2TM in E. coli

[1644] The plasmid for ALG1TM was transferred into and expressed by E. coli BL21 (DE3) according to Rexer et al. (2018) Biotechnol Bioeng 115, 192-205.

[1645] Gene sequences of MGAT1TM and 4GalT1TM were inserted into expression vector pET-28a(+) and gene sequence of MGAT2TM was inserted into expression vector pET-28b(+). Both vectors harbor an N-terminal 6-fold histidine tag (His.sub.6-tag) for purification. Vectors were cloned either in Shuffle T7 Express lysY, for MGAT2TM, or BL21(DE3), for MGAT1TM and 4GalT1TM, competent E. coli cells. Positive transformants, selected by antibiotics, were used for protein expression. In general, cultures were cultivated in TB medium under agitation at 30 C. Overnight cultures were inoculated to an optical density at 600 nm (OD.sub.600) of 0.1. Gene expression was induced at OD.sub.600=0.6 by the addition of 0.4 mM IPTG and cultures were cooled down to 16 C. Cells were harvested after overnight incubation by centrifugation (7,192g, 20 min, 4 C.) and cell pellets were stored at 20 C. until further usage.

1.2 Gene Expression of ALG2 in Saccharomyces cerevisiae and Membrane Fraction Preparation

[1646] ALG2 expression was conducted according to Rexer et al. (2020) J Biotechnol 322, 54-65 using the yeast strain Yeast ORF ALG2 (YGL065C), which was purchased from the Dharmacon Yeast ORF Collection (Cambridge, United Kingdom). For gene expression, 200 mL synthetic drop-out medium without uracil was inoculated with an overnight culture to an OD.sub.600=0.3. Cultivation was performed at 30 C. under agitation at 120 rpm until OD.sub.600 reached 1.2. Gene expression was induced by adding 100 mL YP medium supplemented with 2% galactose (final concentration). Cells were harvested after 23 h of cultivation (120 rpm, 30 C.) by centrifugation (6,000g, 20 min), washed once with ice cold water and cell pellets were stored at 20 C. Cell pellets were resuspended in buffer A (30 mM Tris (pH 7.5), 3 mM MgCl.sub.2, 0.1% IGEPAL) at 1000 bar for 3 cycles. Cell debris was removed by centrifugation for 20 min at 8,000g (4 C.), followed by ultracentrifugation (100,000g for 45 min at 4 C.). Membrane fractions were solubilized in buffer A with 50% (v/v) glycerol.

1.3 Gene Expression of T. brucei STT3A in Sf9 Cells

[1647] STT3A expression and purification were performed according to Ramirez et al. (2017) using flashBAC DNA (Oxford Expression Systems) and Sf9 insect cells (Merck, Darmstadt, Germany) (J Biotechnol 2020, 322, 54-65; Glycobiology 2017, 27, 525-535). A synthetic gene of Trypanosomas brucei coding for STT3A with a 10-fold histidine and YFP tag was purchased from Thermo Fisher Scientific.

1.4 Enzyme Purification

[1648] Enzyme solutions were prepared by cell disruption using a high-pressure homogenizer (3 cycles, 1,000 bar, 4 C.) followed by centrifugation (7,192g, 20 min, 4 C.). Supernatants were then applied to an equilibrated immobilized metal affinity chromatography (IMAC) column using Ni Sepharose HP columns from GE Healthcare (Chicago, USA). Purified enzyme solutions were desalted using Amicon Ultra 0.5 mL Filters from Merck (Darmstadt, Germany) with a molecular weight cut-off of 10 kDa according to the manufacturer's instruction. Desalted enzymes were stored in 50% (v/v) glycerol at 20 C.

2 Glycan Analysis

2.1 CGE-LIF

[1649] The synthesis of LLOs was analyzed by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) (J Proteome Res 2010, 9, 6655-6664; Electrophoresis 2008, 29, 4203-4214; Hennig et al N-Glycosylation Fingerprinting of Viral Glycoproteins by xCGE-LIF in Carbohydrate based vaccines, Methods and Protocols, 1331, 123-143). Glycans were released from the lipid tail by mild acidic hydrolysis using 50 mM HCl for 30 min at 90 C. and neutralized with 100 mM NaOH. Afterwards freeze-dried glycans were labeled with APTS and excess APTS was removed by hydrophilic interaction chromatography with solid phase extraction (HILIC-SPE) (J Proteome Res 2010, 9, 6655-6664). For analysis 1 L sample, 9.6 L HiDi, 0.7 L LIZ base pair standard and 0.7 L 2.sup.nd NormMix were mixed and injected to a 4-capillary DNA sequencer (3130 Genetic Analyzer, Life Technologies, California, USA) with a POP7 polymer matrix (50 cm). Data analysis was carried out using glyXtool.sup.CE (glyXera GmbH, Magdeburg, Germany).

2.2 Mass Spectrometry

[1650] Prior to the analysis by mass spectrometry, enzymes and larger molecules were separated from the reaction products by filtration using a 10 kDa molecular weight cut-off filters (Amicon Ultra 0.5 mL Filters, Merck, Darmstadt, Germany). Samples were desalted by manual C18 chromatography. Therefore, samples were reconstituted in up to 1 mL 0.1% trifluoroacetic acid (TFA) to ensure that pH is lower than 3. HyperSEP C18 columns (ThermoFisher Scientific) were conditioned with 3 mL of conditioning buffer (90% methanol, 10% dH.sub.2O, 0.1% TFA). Prior to sample loading the column was equilibrated with 0.1% TFA in water. Samples were desalted by using 0.1% TFA in water and glycopeptides were eluted with 50% acetonitrile (ACN) in water containing 0.1% TFA. For detergent removal HiPPR detergent removal spin columns (ThermoFisher Scientific) were used according to the manufacturer's instruction. Sample measurement and glycoproteomic analysis was conducted as described in previous studies (Hoffmann et al, 2018 Proteomics 18, e1800282; J Biotechnol 2020, 322, 54-65). Briefly, TAMRA glycopeptides were analyzed by reverse-phase liquid chromatography coupled online to RP-LC-ESI-OT-OT MS/MS (LTQ Orbitrap Elite hybrid mass spectrometer, Thermo Fisher Scientific) using higher-energy collision dissociation fragmentation (HCD) at a normalized collision energy of 20 (NCE 20, HCD.low). Glycopeptide mass spectra were evaluated using Xcalibur Qual Browser 2.2 (Thermo Fisher Scientific) as well as Byonic (Protein Metrics, San Carlos, CA). Relative quantification of TAMRA glycopeptides was performed using Byologic (Protein Metrics).

3 Synthesis of Lipid-Linked Oligosaccharides

[1651] In general, all reactions were performed in a volume of 100 L, at 30 C. and under agitation at 300 rpm. Typically, 10 L aliquots were taken from reaction batches for CGE-LIF analysis.

Example 1: Synthesis of LL-GlcNAc.SUB.2.Man.SUB.3

[1652] The one-pot multi-enzyme run was carried out for 8 h. The reaction batch contained: 50 mM MOPS (pH 6.8), 0.1% IGEPAL, 10 mM MgCl.sub.2 (buffer B), 1 mM DTT, 0.1 mM phytanyl-PP-chitobiose, 2 mM GDP-mannose, 0.1 mg/mL purified ALG1TM (SEQ ID NO 29) and 35% (v/v) ALG2 (SEQ ID NO 30) yeast membrane fraction. After 8 h the reaction batch was quenched for 5 min at 90 C. to ensure enzyme inactivity.

[1653] Results of the xCGE-LIF measurement after a reaction time of 8 h are depicted in FIG. 3. The electropherogram shows a large fraction of the product, LL-GlcNAc.sub.2Man.sub.3 (178 MTU), but also small amounts of LL-GlcNAc.sub.2Man.sub.2 at 140 MTU and LL-GlcNAc.sub.2Man.sub.5 at 248 MTU. Other by-products were not observed in this reaction.

Example 2: Sequential Synthesis of LL-GlcNAc.SUB.2.Man.SUB.3.GlcNAc.SUB.1.Gal.SUB.1.[3]

[1654] For the in vitro synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3] two sequential runs were conducted. The first reaction batch contained 25 mM HEPES (pH7), 0.1% IGEPAL and mM MnCl.sub.2 (buffer C) with 6 mM UDP-GlcNAc, 50% (v/v) LL-GlcNAc.sub.2Man.sub.3 and 15% (v/v) MGAT1TM (SEQ ID NO 26). After 24 h at 30 C., reactions were quenched for 5 min at 90 C.

[1655] FIG. 4 (red) shows that after 24 h a large proportion of LL-GlcNAc.sub.2Man.sub.3 was converted to LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3](217 MTU). Small amounts of intermediates such as LL-GlcNAc.sub.2Man.sub.2 (140 MTU) and LL-GlcNAc.sub.2Man.sub.5 (248 MTU) that originate from the LL-GlcNAc.sub.2Man.sub.3 stock solution were also identified.

[1656] The second reaction batch was conducted in buffer C, 6 mM UDP-Gal, 50% (v/v) LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3] and 25% (v/v) 4GalT1TM. The reaction was performed for 24 h and was quenched at 90 C. for 5 min.

[1657] Results of the reaction run after 24 h are shown in blue in FIG. 4. LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3] from the previous reaction (FIG. 4, red) was fully converted to LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3] and appeared at approximately 260 MTU in the electropherogram. Other products from preceding reaction runs are LL-GlcNAc.sub.2Man.sub.2 (140 MTU) (not shown), LL-GlcNAc.sub.2Man.sub.3 (175 MTU) and LL-GlcNAc.sub.2Man.sub.5 (248 MTU).

Example 3: Stepwise Synthesis of LL-GlcNAc.SUB.2.Man.SUB.3.GlcNAc.SUB.2.Gal.SUB.2

[1658] Sequential synthesis: To synthesize LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2, two reaction runs were carried out sequentially. The first reaction batch contained buffer C with 6 mM UDP-GlcNAc, 50% (v/v) Man.sub.3 and 12.5% (v/v) MGAT1TM and MGAT2TM (SEQ ID NO 27), respectively. After 24 h reactions were quenched at 90 C. for 5 min and centrifuged.

[1659] FIG. 5 (red) shows that after a reaction of 24 hours, a large fraction of LL-GlcNAc.sub.2Man.sub.3 was converted to the lipid-linked complex-type structure LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2. Furthermore, intermediates of the reaction, LL-GlcNAc.sub.2Man.sub.2, LL-GlcNAc.sub.2Man.sub.3 and LL-GlcNAc.sub.2Man.sub.5 were also detected by CGE-LIF analysis.

[1660] The second reaction batch contained: buffer C, 6 mM UDP-Gal, 25% (v/v) 4GalT1TM (SEQ ID NO 28) and 50% (v/v) LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2[3/6]. The reaction was quenched at 90 C. for 5 min after 24 h of incubation. Here (FIG. 5, blue), LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2[3/6] was fully converted to either LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2[3/6](331 MTU) or LL-GlcNAc.sub.2Man.sub.3GlcNAcGal[3](262 MTU). The result demonstrated that the synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 was successful and a large proportion (35%) of the final product was obtained.

[1661] One-pot synthesis: The one-pot synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 was performed in buffer C with 4 mM UDP-GlcNAc, 4 mM UDP-Gal, 35% (v/v) Man.sub.3 and 15% (v/v) MGAT1TM, MGAT2TM and 4GalT1TM, respectively. The reaction was performed for 36 h at 30 C. under agitation (350 rpm). After 0, 4, 8, 24 and 36 h aliquots of 10 L were taken and the reaction was quenched for 5 min at 90 C.

[1662] Samples were analyzed by xCGE-LIF analysis after 4 h, 8 h, 24 h and 36 h. Results of the reaction are depicted in FIG. 6. After 4 h, LL-GlcNAc.sub.2Man.sub.3 was partly converted to LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3]. Within the next 4 h the reaction was driven towards the site product LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3](262 MTU) and the final product LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 (331 MTU). The peak intensity of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2[3/6] after 24 h is slightly higher than after 8 h. After 36 hours, the reaction showed no differences in comparison to the 24 hours sample.

[1663] At the end of the run, the reaction mixture mainly contained LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3](262 MTU). Nevertheless, it was shown that the synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 was successful, even only in a small amount (5%).

Example 4: Transfer of LLOs to Synthetic Peptides by T. brucei STT3A

[1664] To analyze the transfer of LLOs, TAMRA-labeled synthetic peptides with the consensus sequence Asn-Xaa-Thr/Ser (wherein Xaa represents any amino acid except proline) and the following amino acid sequence, G-S-D-A-N-Y-T-Y-T-Q, were purchased from Biomatik (Cambridge, Canada). The reaction was conducted in a volume of 100 L and contained: 20 mM HEPES (pH 7.5), 10 mM MnCl.sub.2, 150 mM NaCl, 0.035% (w/v) DDM, 0.007% (w/v), 50% (v/v) LLO, 20 M synthetic peptides, 36.3% (v/v) T. brucei STT3A (SEQ ID NO 31) and EDTA-free protease inhibitor (Roche, Basel, Switzerland).

[1665] Four one-pot in-vitro glycosylation reactions were conducted, to investigate the ability of recombinant T. brucei STT3A to transfer the complex-type structures LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3] and LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3], LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 and LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2, from the lipid anchor to the asparagine residue of synthetic peptides following the N-glycosylation consensus motif. The transfer was performed using the products of the sequential reactions (see FIGS. 4 and 5) and was analyzed by LC-MS/MS.

[1666] In all runs, the amount of not N-glycosylated or deamidated peptides was approximately 90%. Only glycosylated peptides were considered to determine the relative proportion of transferred glycans (Table 1).

[1667] Non-glycosylated or deamidated peptides were not included. Around 64% of all glycosylated peptides contained a GlcNAc.sub.2Man.sub.5 N-glycan structure. LL-GlcNAc.sub.2Man.sub.5 was present in equal amounts in all reactions due to the naturally occurring ALGT1 in the yeast membrane fraction of recombinant ALG2 (see FIGS. 4 and 5). As LL-GlcNAc.sub.2Man.sub.5 was only present in small amounts, the high proportion of glycosylated Man.sub.5-peptides highlight the high affinity of T. brucei ssOST to LL-GlcNAc.sub.2Man.sub.5. Overall, the transfer of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 was not detected by LC-MS/MS analysis.

TABLE-US-00002 TABLE 1 LC-MS/MS-based N-glycoproteomic analysis of the in vitro N-glycosylation of synthetic and TAMRA-labeled peptides via T. brucei ssOST. The composition and proposed structure of the detected N-glycans present on the TAMRA-labeled peptide are displayed on the left side of the table. In the second and third column the transfer of the sequential synthesis of LL- GlcNAc.sub.2Man.sub.3GlcNAcGal[3] with MGAT1TM and MGAT1TM + 4GalT1TM is shown. The fourth and fifth column show the transfer of reaction products from the sequential synthesis of LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 with MGATTM1/2 and MGATTM1/2 + 4GalT1TM. [] product not in the reaction batch. For each reaction all detected N-glycopeptide are listed with their relative proportion (only glycosylated peptides were considered for the calculation). Modification MGAT1 + [relative proportion MGAT1 + MGAT1 + 2 + in %] MGAT1 B4GALT 2 B4GALT HexNAc(2)Hex(2) [00214] 1.4 0.0 3.1 5.0 HexNAc(2)Hex(3) [00215] 0.0 0.0 5.4 24.2 HexNAc(2)Hex(4) [00216] 7.4 9.4 12.6 7.1 HexNAc(3)Hex(3) [00217] 30.0 HexNAc(2)Hex(5) [00218] 61.1 69.6 60.1 63.7 HexNAc(3)Hex(4) [00219] 21.1 0.0 HexNAc(4)Hex(3) [00220] 18.8

[1668] In the first run, MGAT1TM was used to synthesize LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3]. After the subsequent in vitro glycosylation reaction using ssOST, the extracted ion chromatograms (EIS-MS) of identified glycopeptide spectra are depicted in FIG. 7.

[1669] Peptides with GlcNAc.sub.2Man.sub.2, GlcNAc.sub.2Man.sub.4, GlcNAc.sub.2Man.sub.5 and GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3]N-glycan structures were identified. The fraction of glycopeptides containing GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3] was 30% (see Table 1). In the second run, the addition of MGAT1TM and 4GalT1TM was used to generate LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3]. GlcNAc.sub.2Man.sub.4, GlcNAc.sub.2Man.sub.5 and GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3]N-glycan structures were identified by LC-MS/MS measurement (FIG. 7B). The in vitro glycosylation reaction yielded the GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3]-peptide with a proportion of 21.1% (Table 1). N-glycopeptides containing GlcNAc.sub.2Man.sub.3GlcNAc.sub.1[3] were not detected by LC-MS/MS measurement. This finding is consistent with the LLO synthesis reaction run (see FIG. 4). There, the side product LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.1Gal.sub.1[3] was not detected.

[1670] In the third run, MGAT1TM and MGAT2TM were applied to generate LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 first, followed by using the ssOST to glycosylate the target peptide. The transfer of complex-type GlcNAc.sub.2Man.sub.3GlcNAc.sub.2 to the synthetic peptide was successful (see FIG. 7C and Table 1). Also, side-products such as peptides containing GlcNAc.sub.2Man.sub.2, GlcNAc.sub.2Man.sub.3, GlcNAc.sub.2Man.sub.4 and GlcNAc.sub.2Man.sub.5 were identified. The relative proportion of the GlcNAc.sub.2Man.sub.3GlcNAc.sub.2-peptide was 18.8% of all glycosylated peptides in this reaction.

[1671] In the fourth run, after synthesizing LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2, an in vitro reaction was initiated. The results show that this LLO is not transferred by the ssOST to synthetic peptides (FIG. 7D & Table 1). Only the side-products GlcNAc.sub.2Man.sub.2, GlcNAc.sub.2Man.sub.3, GlcNAc.sub.2Man.sub.4 and GlcNAc.sub.2Man.sub.5 were identified.

Example 5: Transfer of High-Mannose LLOs to a Synthetic Influenza Hemaglutinin Peptide by T. brucei STT3A

[1672] The reaction was conducted in a final volume of 50 L containing reaction buffer (20 mM HEPES, 10 mM MnCl.sub.2, 150 mM NaCl, 0.035% (w/v) DDM, 0.007% (w/v) CHS and protease inhibitor), 25% (v/v) LL-GlcNAc.sub.2Man.sub.3/Man.sub.5, 0.02 mM peptide and 20 nM OST. The OST was added step-wise at the beginning, after 4 h and 8 h of the reaction. The reaction was stopped after 24 h by heat (10 min, 90 C.). Afterwards, a Tris-Tricine PAGE was performed and analyzed using a fluorescence scanner.

[1673] The peptide sequence was derived from Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) Hemaglutinin (Uniprot Number: P03452; Amino acids 29-49) (SEQ ID NO: 2): TAMRA-STDTVDTVLEKNVTVTHSVNL-NH.sub.2 The peptide was synthesized and purchased from Biomatik.

Example 6: In Vitro N-Glycosylation of HA1 Protein Having 100 Amino Acids by Recombinant T. brucei STT3A

[1674] The in vitro N-glycosylation was performed under the conditions as described in the previous example. The peptide sequence was derived from Influenza A virus (strain A/Puerto Rico/8/1934 H1N1) Hemaglutinin (Uniprot Number: P03452; Amino acids 29-49) (SEQ ID NO: 32):

TABLE-US-00003 TAMRA- STDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGW LLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVS SF-NH.sub.2

[1675] The peptide was synthesized and purchased from Biomatik.

[1676] The reaction mixture was analyzed by in-gel PNGase F digestion followed by CGE-LIF analysis (see FIG. 9).

Example 7: In Vitro Glycoengineering of HA1 Protein Having 100 Amino Acids

[1677] The Man.sub.3/Man.sub.3-HA1 glycoprotein obtained in Example 6 was stepwise converted to various complex glycan structures using recombinant transmembrane-deleted glycosyltransferases and sugar nucleotides.

[1678] In a first step, UDP-GlcNAc and the enzymes MGAT1TM & MGAT2TM were brought in contact with Man.sub.3/Man.sub.3-HA1 glycoprotein under glycosylation reaction conditions as described in the previous examples. Analysis of the reaction mixture by in-gel PNGase F digestion followed by CGE-LIF analysis revealed the formation of GlcNAc.sub.2Man.sub.3GlcNAc-peptide and GlcNAc.sub.2Man.sub.3GlcNAc.sub.2-peptide (see FIG. 10).

[1679] Subsequently, the glycoprotein was subjected to a galactosylation reaction by UDP-galactose in the presence of recombinant 4GalT1TM enzyme. Analysis of the reaction mixture by in-gel PNGase F digestion followed by CGE-LIF analysis revealed the formation of GlcNAc.sub.2Man.sub.3GlcNAcGal-peptide and GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2-peptide (see FIG. 11).

[1680] In a third step, the glycoprotein was subjected to a sialylation reaction by CMP-Neu5Ac in the presence of recombinant recombinant St6Gal1TM enzyme. Analysis of the reaction mixture by in-gel PNGase F digestion followed by CGE-LIF analysis revealed the formation of GlcNAc.sub.2Man.sub.3GlcNAcGalNeu5Ac-peptide and GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2Neu5Ac.sub.2-peptide (see FIG. 12).

Example 8: In Vitro Glycoengineering of Lipid-Linked Oligosaccharides (LLO)s

[1681] Lipid-linked Man.sub.3 (as obtained in Example 1) was subjected to a glycosylation cascade reaction with UDP-GlcNAc, UDP-Gal in the presence of recombinant MGAT1, MGAT2 & MGAT5 enzymes followed by the addition of 4GalT1 enzyme. The reaction was carried out under conditions as described in Example 2. Analysis of the reaction mixture by in-gel PNGase F digestion followed by CGE-LIF analysis revealed the formation of LL-GlcNAc.sub.2Man.sub.3GlcNAcGal, LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.2Gal.sub.2 and LL-GlcNAc.sub.2Man.sub.3GlcNAc.sub.3Gal.sub.3 (see FIG. 13).

Example 9: In Vitro N-Glycosylation of HA1 Protein Having 100 Amino Acids Using Recombinant STT3A

Description:

[1682] Transferring LL-Man.sub.3 to the N-glycosylation sequence of the synthetic peptide. Subsequently generating a complex glycan structure A2G2S2 with terminal sialic acid by applying the recombinant enzymes MGAT1, MGAT2, a galactosyltransferase and a sialyltransferase along the activated sugars UDP-GlcNAc, UDP-galactose and CMP-Neu5Ac.

Materials:

[1683] 100 amino acid protein purchased from Biocat/Biomatik with the following amino acid sequence SEQ ID NO: 32):

TABLE-US-00004 (STDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAG WLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSV SSF) [1684] .fwdarw.glycosylation site is labeled in bold. [1685] Reactions conditions: As described in previous examples.

Analytics:

[1686] In-gel PNGase F digestion followed by CGE-LIF analysis

Results:

[1687] See, FIGS. 14-17.

Example 10: Synthesis of Engineered LLOs by Commercial Enzymes

Description:

[1688] Generating non-natural lipid-linked glycans as substrate for in-vitro glycosylation reactions. The lipid carrier is phythanyl. LL-Man.sub.3 was treated with the recombinant glycosyltransferases MGAT1, MGAT2 (both in-house produced) and MGAT5 (commercial) along with UDP-GlcNAc. After incubation, b4GalT (in-house produced) was added along UDP-Gal.

Materials:

[1689] HEPES buffer, pH 6.8 [1690] MGAT1, MGAT2, MGAT5, b4GalT [1691] UDP-GlcNAc, UDP-Gal [1692] LLO mixture containing LL-Man3 [1693] Reactions conditions: 30 C., 500 rpm, 24 h incubation each

Analytics:

[1694] Mild acid hydrolysis followed by CGE-LIF analysis

Results:

[1695] See, FIGS. 18-19.

TABLE-US-00005 TABLE 2 Sequence Listing SEQ ID No Name 1 synthetic peptide with consensus sequence 2 synthetic influenza hemaglutinin peptide 3 Campylobacter jejuni strain RM1221 oligosaccharyltransferase enzyme 4 Campylobacter lari strain RM2100/D67/ATC BAA-1060 oligosaccharyltransferase enzyme 5 Trypanosoma brucei oligosaccharyltransferase enzyme 6 Homo sapiens oligosaccharyltransferase enzyme 7 Mus musculus oligosaccharyltransferase enzyme 8 Canis lupus familiaris oligosaccharyltransferase enzyme 9 Canis lupus familiaris oligosaccharyltransferase enzyme 10 Caenorhabditis elegans oligosaccharyltransferase enzyme 11 Bos taurus oligosaccharyltransferase enzyme 12 Pongo pygmaeus abelii oligosaccharyltransferase enzyme 13 Oryza stiva japonica oligosaccharyltransferase enzyme 14 Oryza stiva japonica oligosaccharyltransferase enzyme 15 Dictyostelium discoideum oligosaccharyltransferase enzyme 16 Brachypodium distachyon oligosaccharyltransferase enzyme 17 Brachypodium retusum oligosaccharyltransferase enzyme 18 Rattus norvegicus oligosaccharyltransferase enzyme 19 Brachypodium sylvaticum oligosaccharyltransferase enzyme 20 Brachypodium pinnatum oligosaccharyltransferase enzyme 21 Brachypodium rupestre oligosaccharyltransferase enzyme 22 Saccharomyces cerevisiae strain ATCC 204508/S288c oligosaccharyltransferase enzyme 23 Schizosaccharomyces pombe strain 972/ATCC 24843 oligosaccharyltransferase enzyme 24 Arabidopsis thaliana oligosaccharyltransferase enzyme 25 Arabidopsis thaliana oligosaccharyltransferase enzyme 26 transmembrane domaine deleted alpha-1,3-mannosyl- glycoprotein 2-beta-N-acetylglucosaminyltransferase 27 transmembrane domaine deleted alpha-1,6-mannosyl- glycoprotein 2-beta-N-acetylglucosaminyltransferase 28 transmembrane domaine deleted beta-1,4-galactosyltransferase 1 29 beta-1,4-mannosyltransferase 30 alpha-1,3/1,6-mannosyltransferase 31 STT3A oligosaccharyltransferase from T. brucei 32 synthetic peptide with consensus sequence