1. Production and Glycosylation Analysis of Model Proteins from a Vaccinia Virus-Mammalian Cell Expression System Nicole A. Bleckwenn a,b,c, William Bentley b,c, and Joseph Shiloach a AbstractBackground Expression Verification Defining the Culture System Conclusions A vaccinia virus-mammalian cell expression system was developed as an alternative method for recombinant protein production utilizing EGFP as a reporter protein (1). In previous work, EGFP production was evaluated in T-flask culture and in both suspension and microcarrier based bioreactor systems, where general production parameters were defined. In this work, the production capability of the system, as defined with EGFP, was evaluated using two proteins, the HIV gp120 envelope glycoprotein and hGC-1 (2), an olfactomedin-related protein. These proteins contain complex post-translational modifications, required for gp120 activity and possibly for hGC-1, although there is little information on this recently discovered protein. Two recombinant vaccinia virus strains were engineered with the genes for gp120 or hGC-1 and e xpression of these proteins was achieved in either T-flask culture or both T-flask and bioreactor culture by infection of the cell culture with recombinant virus. The production process, purification protocol and glycosylation pattern of gp120 is described. Bioreactor culture produced secreted gp120 up to 40 mg/L at 66 hours post infection (hpi). a Biotechnology Unit, NIDDK, National Institutes of Health, DHHS, Bethesda, MD b Center for Biosystems Research, UMBI, College Park, MD c Department of Chemical Engineering, UMCP, College Park, MD Production Parameters gp120-6xHis Productiongp120-6xHis Purification Deglycosylation Analysis Purification Methods Deglycosylation Results References Based on reporter protein (EGFP) process development Cell Growth 5 g/L Cytodex 3 microcarriers 1.5x10 5 HeLa cell/mL initial seeding DMEM+10% fetal bovine serum 37°C, 30% dissolved oxygen, pH 7.0 Growth for 5 days to 1-2x10 6 cell/mL Infection 1 hour duration MOI 5.0 (pfu/cell) One third working volume No serum in infection media (DMEM) 1 mM IPTG added at infection Production Return to 1.5 L volume and 10% serum Reduce temperature to 34°C Increase dissolved oxygen to 50% 66 hours post infection Clarify by centrifugation 48 hpi Reactor Supernatant Load column with 50% slurry Ni-NTA resin (Qiagen) 1 mL resin per 100 mL supernatant Wash column with base buffer (50 mM NaH 2 PO 4 300 mM NaCl pH 8.0) Bind protein Recycle supernatant through column for 4 hours Wash column 1X with base buffer 2X with base buffer + 20 mM imidazole Elute protein Four fractions with base buffer + 200 mM imidazole Treat to deactivate virus 0.5% NP-40 500 mM NaH 2 PO 4 300 mM NaCl Incubate overnight 4°C Vaccinia Virus Orthopoxvirus family Poxviridae Transcription occurs in cytoplasm of infected cell Wide host range, includes most mammalian species and humans HeLa cells Attachment dependent strain (ATCC CCL-2) Microcarrier growth for larger cultures Enhanced Green Fluorescent Protein (EGFP) Used as reporter protein to develop system parameters hGC-1 Olfactomedin-related protein (~64 kDa) Potential for treatment of prostate and other cancers Six N-linked glycosylation sites gp120 HIV envelope coat protein (~120 kDa) Almost half the molecular weight attributed to N-linked glycosylations which are required for activity of the protein (3) 100 mg/L gp120 10 mg/L gp120 5 mg/L gp1201 mg/L gp120 Prior to purification Flow through Wash 1Wash 2Wash 3 Elution 1Elution 2Elution 3Elution 4 100 mg/L gp120 10 mg/L gp120 5 mg/L gp1201 mg/L gp120 Prior to purification Flow through Wash 1Wash 2Wash 3 Elution 1Elution 2Elution 3Elution 4 Coomassie Stained Gel Western Blot 250 148 60 42 30 22 17 6 4 kDa Virus Construction vT7lacOI * virus HeLa Cells Transfect Infect Plaque isolation Amplification Viral purification Viral titer Donor Plasmid Gene pVOTE.2 * Plasmid Gene Recombinant Plasmid Recombinant virus * vT7lacOI virus and pVOTE.2 plasmid were kindly supplied by B. Moss and P. Earl, NIAID, NIH (4) and gp120 plasmid pTM-DHgp120H was kindly provided by M. Cho, School of Medicine, Case Western Reserve University (5). Vacuum Level Control Feed Pump Water Jacket Inlet Outlet DO pH Agit. Temp. Filtrate Pump Air Inlet Mesh Screen Module Diaphragm ATF Controller Gas inlet into reactor headspace HeLa cells (attachment dependent) Microcarrier perfusion culture 1.5 L working volume reactor ATF™ System *** for microcarrier perfusion culture *** ATF™ System was kindly supplied by Refine Technology, Co., East Hanover, NJ. 1. Bleckwenn, N.A., W.E. Bentley, and J. Shiloach, Biotechnology Progress, 2003. 19(1): p. 130-136. 2. Zhang, J.C., et al., Gene, 2002. 283(1-2): p. 83-93. 3. Hu, Y.C., et al., Biotechnology Progress, 2000. 16(5): p. 744-750. 4. Ward, G.A., et al., Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(15): p. 6773-6777. 5. Lee, M.K., M.A. Martin, and M.W. Cho, Aids Research and Human Retroviruses, 2000. 16(8): p. 765-775. Three different proteins were produced from three constructed viruses (EGFP, hGC-1, and gp120) Growth, infection and production parameters were defined using reporter protein EGFP gp120 was produced in a 1.5L bioreactor culture over 8 days with production for 66 hours Per liter of working volume 4.7 mg secreted and purified gp120 was recovered at 48 hpi 4 liters of media used 200 mL serum used 370  L viral stock used (1.9x10 10 pfu/mL) Deglycosylation analysis of gp120 showed significant amount of N- linked glycosylations of approximately half the mass of the protein Similar analysis will be performed on bioreactor culture with hGC-1 containing virus EGFP Reporter Protein (intracellular) hGC-1 Protein (intracellular) gp120 Protein (extracellular) Anti-FLAG antibody 250 148 60 42 30 22 17 6 4 kDa Negative control + hGC-1-FLAG gene + EGFP-6xHis gene Induced + EGFP-6xHis gene Not Induced No virus Fluorescent Microscopy Images ** Anti-gp160 Antibody, HIV-1 gp160 Antiserum (HT3) from DAIDS, NIAID, NIH produced under contract by Repligen was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Western Blots Anti-gp160 ** antibody Negative control + gp120-6xHis gene 250 148 60 42 30 22 17 6 4 kDa Cleavage of N-linked Sugars PNGase F – Cleaves most N-linked oligosaccharides (unless  (1-3) core fucosylated) Cleavage of Common O-linked Sugars O-Glycosidase – Cleaves O-linked unsubstituted Gal-  (1-3)- GalNAc-  -  -2(3,6,8,9) Neuraminidase – Cleaves non-reducing terminal branched and unbranched sialic acids Cleavage of less common hexasaccharide structures  (1-4)Galactosidase – Cleaves  (1-4)-linked, non-reducing terminal galactose  -N-Acetylglucosaminidase – Cleaves  -linked N- acetylglucosamine gp120 purified from reactor at 48 hpi in 10 mM Tris pH 7.0 Aliquot five fractions and treat sequentially with enzymes 12345 Treatment Quantification achieved by scanning blot and comparing to gp120 standard **** with Scion Image software, Scion Corp. 1866524844403024Time (hpi) Western blot bands **** gp120 standard, HIV-1 SF162 gp120 from Chiron Corporation and the DAIDS was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. 1.4x10 6 cell/mL infected at 0 hpi gp120 standard Treatment 1Treatment 2Treatment 3Treatment 4 Treatment 1 – No enzymes Treatment 2 – PNGase F Treatment 3 – PNGase F O-Glycosidase Treatment 4 - PNGase F O-Glycosidase  -2(3,6,8,9) Neuraminidase Treatment 5 - PNGase F O-Glycosidase  -2(3,6,8,9) Neuraminidase  (1-4)Galactosidase  -N-Acetylglucosaminidase Treatment 5 250 148 60 42 30 22 17 6 4 kDa Std