Mammalian-type Glycosylation l in LEXSY Case Study: Recombinant hu Erythropoietin Jena Bioscience GmbH Loebstedter Str. 80 07749 Jena, Germany Tel.: +49-3641-628-5000 Fax: +49-3641-628-5100 628 e-mail: info@jenabioscience.com
Hu EPO was secreted and N-glycosylated in LEXSY 1 2 3 4 5 6 7 80 49 35 29 1 no enzyme 2 N-glycosidase 3 O-glycosidase 4 neuraminidase 5 enzyme mix 6 mature intracell. EPO 7 negative control (host strain) 20 Deglycosylation of recombinant hu erythropoietin (EPO) expressed in LEXSY with various glycosidases 2
Hu EPO purified from LEXSY was natively processed Ammonium sulfate precipitation of culture supernatants N-terminal aa sequencing Concanavalin A affinity chromatographie APPRLIC... Phenyl Sepharose Native processing of EPO signal peptide HSA Signal peptide MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLQRYLLE FTAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAV SDS-PAGE silver stained EPO EVWQGLALLSEAVLRGFTQALLVNSSQPWEPLQLHVDKAVSGLR SLTTLLRALGAQKEAISPPDAASAAPLRTITADFTTFRKLFRVYSNF LRGKLKLYTGEACRTGDR 3
Hu EPO was modified to only two glycoforms in LEXSY Gal β1 4 GlcNAc β1 2 Man α1 Gal β1 4 GlcNAc β1 2 Man α1 Fuc α1 6 6 Man β1 4 GlcNAc β1 4 GlcNAc 3 complex Man α1 Man α1 6 3 Man β1 4 GlcNAc β1 4 GlcNAc core vs. multiple glycoforms in CHO Enzymatic glycan analysis 4
LEXSY N-Glycosylation is of mammalian type ( ) ( ) +3rd +4th antenna Bacteria Yeast e.g. Pichia Insect cells e.g. Sf9/21 Leishmania tarentolae determined with EPO, IFNγ & GP63 Mammalian cells Galactose N-acetylneuraminic acid LEXSY Mannose Fucose N-acetylglucosamine Polypeptide 5
rhu EPO from LEXSY was biologically fully active NC PC CFU-GEMM* cell proliferation assay hu CD34 + peripheral blood stem cells 5 U EPO (CHO) Dose-dependent proliferation and differentiation in haemoglobin producing cells 1U 5U 10 U 065U 0.65 13U 1.3 36U 3.6 Native signal peptide 1.2 x 10 5 U/mg Leishmania signal peptide 4.0 x 10 5 U/mg * Granulocytes-erythrocytes-monocytes-macrophages y y y 6
Exceptional homogeneity of N-glycosylation in LEXSY Recombinant hu EPO from LEXSY was biologically fully active natively processed at the N-terminus mammalian-type N-glycosylated The N-glycosylation profile was exceptionally homogenous with a biantennary fully galactosylated Man 3 GlcNAc 2 core-α-1,6-fucosylated structure Human recombinant EPO was exceptional homogenously N-glycosylated A: homogenously N-glycosylated EPO from LEXSY B: N-deglycosylated EPO from A C A B C: heterogenously glycosylated EPO from CHO Exceptional homogeneity of N-glycosylation will be of advantage for structure analysis of glycosylated recombinant proteins isolated from LEXSY! Ref. Breitling R, Klingner S, Callewaert N, Pietrucha R, Geyer A, Ehrlich G, Hartung R, Müller A, Contreras R, Beverley S and Alexandrov K (2002) Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. ProteinExpression and Purification 25: 209-218.
Part 2 Glycosylation was demonstrated as well with other secretory eto target proteins 8
Glycosylation of secretory target proteins was inhibited in vivo by addition of Tunicamycin H P1 P1 P2i P2i P2i MW P3i P3i P3i 118 85 47 H LEXSY host (negative control) MW prestained molecular size marker P1 constitutive secretory expression P2i inducible secretory expression P3i inducible secretory expression (enzymatic deglycosylation was shown, slide 10) Tm Tunicamycin added to culture at 10 μg/ml 36 26 Glycosylated target protein from LEXSY Non-glycosylated target protein from LEXSY Tm - - + - - + - + - Concentrated culture supernatants of LEXSY clones Mobility shift caused by addition of Tunicamycin to culture indicated glycosylation l of targett proteins in LEXSY 9
In vitro deglycosylation of LEXSY expressed protein Target protein was affinity-purified from culture medium and enzymatically deglycosylated 1 2 3 4 5 6 7 118 85 1 imidazol eluted POI 2 concentrated POI in PBS 3 prestained molecular size marker 4 N-Glycosidase F (138 ng = 250U) 36 kda 5 POI in deglycosylation buffer 6 POI + N-Glycosidase F 7 imidazol eluted POI POI: target protein of interest 47 36 2 glycoforms consistent with previous findings (EPO) shift to one band Ref. to slide 4, lane P3i 10
Appendix Addition of Tunicamycin affects growth and vitality of L. tarentolae 10 nm Growth 600 1 0 Tm 10 Tm 20 Tm 30 Tm 40 Tm 0,1 0 22 44 68 Optimal concentration for in vivo Tunicamycin inhibition of glycosylation was 10 μg/ml 11