Browsing by Subject "recombinant protein"
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Item Downstream Processing of Recombinant Proteins from Transgenic Plant Systems: Phenolic Compounds Removal from Monoclonal Antibody Expressing Lemna minor and Purification of Recombinant Bovine Lysozyme from Sugarcane(2012-07-16) Barros, GeorgiaTransgenic plant systems have been proposed as bioreactors in the production of pharmaceutical and industrial proteins. The economic benefits of inexpensive plant production systems could be erased if the downstream processing ends up being expensive. To avoid monoclonal antibody (mAb) modification or fouling of chromatography resins, removal of phenolics from plant extracts is desirable. Removal of major phenolics in Lemna extracts was evaluated by adsorption to PVPP, XAD-4, IRA-402 and Q-Sepharose resins. Analysis of phenolics adsorption to XAD-4, IRA-402 and Q-Sepharose showed superior dynamic binding capacities at pH 4.5 than at 7.5. The economic analysis using SuperPro Designer 7.0 indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The overall mAb processing cost can be reduced by implementing a phenolics removal step. To understand phenolics-resin interactions, adsorption isotherms of phenolic compounds (chlorogenic acid, ferulic acid, rutin, syringic acid and vitexin-2-O-rhamnoside) from different phenolic classes on three resins (IRA-402, PVPP, XAD-4) at pH 4.5 and 7.5 were determined. Differences in adsorption with the type of phenolics were observed, and PVPP was not efficient for phenolics removal. Screening of sugarcane lines for bovine lysozyme (BvLz) accumulation indicated that expression levels are still inadequate for commercial development. To maximize BvLz extraction, pH and ionic strength were evaluated; five conditions resulted in equivalent BvLz/TSP ratio. Membrane filtration process using BvLz extracts attained partial removal of native proteins by the 100 kDa membrane step, but also BvLz loss (21-29%). Regardless of the extraction condition, at least 47% of the starting BvLz was lost during the membrane processing. None of the evaluated extraction conditions caused a substantial recovery of BvLz in the concentrate. Alternative purification options for the IEX+HIC process, which achieved 95% BvLz purity, were tested. Direct loading of sugarcane extract concentrate on HIC and XAD-4 pretreatment of juice did not recovered BvLz as effectively as the IEX chromatography. Pure BvLz was obtained by the XAD+HIC process, but higher purification fold and HIC yield were achieved by the IEX+HIC process, due to the complete separation of BvLz and 18-kDa protein.Item Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15(2013-11-21) Burns, Gregory WillisAfter 40 years of research, in vitro systems for mammalian embryo production produce lower quality embryos than those derived from in vivo sources. Recent reports have demonstrated that in vitro bovine oocyte maturation systems benefit from the addition of oocyte secreted factors, specifically Bone Morphogenic Protein 15 (BMP15) from heterologous sources. However, known amino acid sequence variation and species-specific patterns of post-translational glycosylation lead us to hypothesize that utilization of bovine-specific oocyte secreted factors would be more beneficial than the observed effects of heterologous factors. To test this hypothesis, wild type, bovine BMP15 was cloned using reverse transcriptase PCR from RNA obtained from bovine ovarian tissue. For improved detection and purification of the biologically active recombinant protein, a FLAG tag peptide sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was incorporated into the wild type BMP15 gene by PCR. This modified protein was cloned into the pCDNA 3 mammalian expression vector. HEK-293 (human embryonic kidney 293) and FBK (fetal bovine kidney) cell lines were transfected via electroporation and then selected to homogeneity. Collection and purification of rbFL-BMP15 from conditioned medium was accomplished by incubation with anti-FLAG affinity gel and the use of 3X FLAG peptide for elution. Peptides of 15.4 kDa and 17 kDa were noted from the human HEK-293 transfected cell line, while in contrast, bovine FBK cells produced a single 17 kDa protein. Bioactivity and BMP receptor signaling specificity were ascertained using in vitro treatment of HeLa cells and Western blotting for the BMP signaling molecule phosphorylated-SMAD 1/5. Inhibition of this signaling cascade using dorsomorphin, a selective bone morphogenic protein receptor I inhibitor, demonstrated the purified proteins served as BMP15-like agonists. To examine the impact of our purified, bovine-specific peptides on oocyte maturation, cumulus oocyte complexes were in vitro matured for 24 hours in the presence of 100 ng/ml recombinant human BMP15 or rbFL-BMP15 from human or bovine cell lines. Real time quantitative PCR analysis of BMP15 stimulated genes, PTGS2 and TSG6, revealed statistically significant increases in transcript level for treatment with human BMP15 by a Dunnett?s test (p<0.05). In this report, however, we failed to detect a significant affect of rbFL-BMP15 on the gene expression of in vitro mature cumulus oocyte complexes at 24 hours with 100 ng/ml rbFL-BMP15. Future studies including differing time points and concentrations, along with the addition of GDF9 to form a possible heterodimer should be investigated for the possibility of improving bovine oocyte in vitro maturation.