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Transgenic 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.

Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, serves as a model organism to study the circadian clock in plants and animals. Rhodopsins are blue/green-light photoreceptors also found in C. reinhardtii. Chlamyrhodopsin (COP), the most abundant eyespot protein, was reported to have no role in the phototactic and photophobic responses in C. reinhardtii. Its function is yet unknown. In the present study, we hypothesized that the function of COP is to mediate entrainment of the circadian clock by light. In order to test this hypothesis, a C. reinhardtii selection marker conferring resistance to the antibiotic paromomycin was cloned into a COP RNAi construct obtained from another lab. Firstly, the COP RNAi construct was restriction digested to linearize it. The linearized plasmid was then blunt ended with T4 DNA polymerase and dephosphorylated with phosphatase. The linearized fragment was ligated with the paromomycin resistance marker obtained by restriction digestion of the plasmid containing it and transformed into E.coli. The recombinant clones obtained were confirmed by restriction digests. Fusion regions and the orientation of the insert in the recombinant plasmid were confirmed by sequencing. An attempt was made to transform C. reinhardtii with the construct, but this was not successful. Future studies will be required to optimize the C. reinhardtii transformation method. Transformants with reduced COP amounts can then be tested for defects in resetting the clock after light pulses. This will determine whether chlamyrhodopsin is involved in the circadian input pathway or not. The results of the complete project are expected to contribute to our understanding of the circadian clock of many other organisms including humans.

Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.

In this study, effects of different carbon sources and their feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris were investigated by means of cell growth, recombinant protein production and expression levels of hGH and alcohol oxidase (AOX) genes. In this content, firstly, the strain to be used for high level rhGH production was selected between the two phenotypes, i.e., P. pastoris hGH-Mut+ and P. pastoris hGH-MutS. In this selection both phenotypes were compared in two different media containing glycerol/methanol or sorbitol/methanol and P. pastoris-hGH-Mut+ strain grown on medium containing 30 g/L sorbitol with 1% (v/v) methanol was found to have the highest hGH expression level and rhGH production level, 9.84x109 copies/mg CDW and 120 mg/L, respectively. Thereafter, effects of sorbitol, mannitol, fructose, lactose, sucrose, citric acid, lactic acid and acetic acid were investigated by using P. pastoris hGH-Mut+ strain in laboratory scale bioreactors. Among them sorbitol and sucrose were selected to be compared for production in pilot scale bioreactors by adding them batch-wise at the beginning of induction phase with fed batch methanol feeding scheme at &amp / #956 / =0.03h-1. It was shown that sucrose does not support cell growth as sorbitol although it does not repress recombinant protein production. Then three different feeding strategies were applied to develop sorbitol/methanol mixed feeding i) single sorbitol addition at t=0, ii) besides at t=0, adding second batch-wise sorbitol at t=9 h, iii) giving pulse methanol at t=24 h to trigger AOX promoter. These three strategies were compared with a production without addition of co-substrate sorbitol. Substrate consumption, cell growth, recombinant protein production and expression levels of hGH and AOX were investigated for these different feeding strategies. The highest cell concentration was achieved in third strategy as 55 g/L where the highest extracellular rhGH production (301 mg/L) was achieved in the second strategy, with addition of two times of sorbitol. For this highest recombinant protein production case, overall cell and product yield on total substrate were found as 0.17 g/g and 1.71 mg/g, respectively. Moreover, the highest hGH and AOX expression levels were obtained in this strategy.

In this study, resolution of racemic mandelic acid by enyzme enhanced ultrafiltration (EEUF) was studied. In order to develop a methodology, bovine serum albumin (BSA) was used as a model protein for polymer enhanced ultrafiltration (PEUF) experiments and the enzyme S-mandelate dehydrogenase was used for EEUF experiments. To be used for enzyme enhanced ultrafiltration experiments, the gene which is responsible from the production of S-mandelate dehydrogenase was isolated from Pseudomonas putida, expressed in Escherichia coli and the recombinant enzyme was produced. For PEUF experiments, effects of pH and ligand ratio were investigated. Total retention of mandelic acid increased with decrease in pH and total retention of mandelic acid reached to a maximum value of 74.4% at pH 4.3. For EEUF experiments, pH and ligand ratio effect on total retention, enantiomeric excess, enantioselectivity were investigated. Although apoenzyme was tried to be obtained by diafiltration and conversion was tried to be prevented, conversion occured especially at high pH values. To create the apoenzyme effect, three methods were studied. Enzyme conversion was prevented by sodium sulfite inhibition but enzyme did not retain mandelic acid. By oxygen saturation of enzyme, conversion was prevented, binding was achieved but enzyme showed no enantioselectivity. When the enzyme was diafiltrated at pH 10.0, total mandelic acid retention, enantiomeric excess and enantioselectivity reached to 77.2%, 38.9%, 2.27, respectively and the enzyme selectivity was reversed as R-selective.

Stem rust and Yellow rust diseases are the two major wheat fungal diseases causing considerable yield losses in Turkey and all around the world. There are studies which are carried out to identify and utilize resistance sources in order to obtain resistant lines of wheat. However, virulent pathotypes are continuously being important threats to wheat production and yield. For that reason, new approaches for rapid identification are needed. The aim of this study was to investigate and to understand the structural and functional differences between the resistant and sensitive durum wheat cultivars to the plant fungal diseases of stem and yellow (stripe) rusts. To aim this, forty durum wheat recombinant inbred lines (RILs), which were previously determined to be resistant or sensitive to stem and yellow rust diseases, were investigated by the noninvasive Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Also, classification of the resistant and sensitive lines depending on the structural and functional differences has been attempted. The FTIR spectra for stem rust disease showed that, resistant durum wheat lines had a significant increase in the population of unsaturation in acyl chains of lipid molecules, an increase in lipid and in total protein content and also an increase in carboxylic acids and alcohols. For yellow rust disease, resistant lines had a significant increase in hydrogen bonding and they had also a more ordered membrane structure. In Principal Component Analysis for stem rust disease, according to 3700-650 cm-1 region, amide III band (1213-1273 cm-1 region) and C-H stretching region (3020- 2800 cm-1), the resistant and sensitive groups were separated successfully. For yellow rust disease, according to 3700-650 cm-1 region, Amide A and Amide III bands, the resistant and sensitive lines were grouped distinctly. FTIR spectroscopy provides a useful approach to determine the differences in molecular structure of durum wheat RILs regarding resistance of lines to fungal diseases. However, further research is still needed to ensure if the structural and functional differences in biomolecules of the samples could be used as molecular markers for discrimination of rust resistant materials from rust sensitive ones.

No description available.

<p>The aim of this work was to investigate and determine central parameters that can be used to control and increase the solubility, quality and productivity of recombinant proteins. These central parameters should be applicable under the constraints of high-throughput protein production in <em>Escherichia coli.</em></p><p>The present investigation shows that alternative methods exist to improve solubility, quality and productivity of the recombinant protein. The hypothesis is that by reducing the synthesis rate of the recombinant protein, a higher quality protein should be produced. The feed rate of glucose can be used to decrease the synthesis rate of the recombinant protein.</p><p>The influence of feed rate on solubility and proteolysis was investigated using the <em>lac</em>UV5-promoter and two model proteins, Zb-MalE and Zb-MalE31. Zb-MalE31 is a mutated form of Zb-MalE that contains two different amino acids. These altered amino acids greatly affect the solubility of the protein. The soluble fraction is generally twice as high using Zb-MalE compared to Zb-MalE31. Using a low feed rate compared to high benefits the formation of the full-length soluble protein. Furthermore, by using a low feed rate, the proteolysis can be decreased. One other factor that influences the solubility is the amount of inducer used. An increase from 100 µM to 300 µM IPTG only results in more inclusion bodies being formed, the fraction of soluble protein is the same.</p><p>The quality aspect of protein production was investigated for a secreted version of Zb-MalE using two different feed rates of glucose and the maltose induced promoter P<em>malK</em>. It was shown that when the protein was secreted to the periplasm, the stringent response as well as the accumulation of acetic acid (even for high feed rates) was reduced. The stringent response and accumulation of acetic acid are factors that are known to affect the quality and quantity of recombinant proteins. Transporting the protein to the periplasm results in this case on a lower burden on the cell, which leads to less degradation products being formed when the protein is secreted to the periplasm.</p><p>Seeing the feed rate as a critical parameter, the high-throughput production would benefit from a variation in the feed rate. However, since the fed-batch technique is technically complicated for small volumes another approach is needed. <em>E.coli</em> strains that have been mutated to create an internal growth limitation that simulate fed-batch were cultivated in batch and were compared to the parent strain. It was shown that the growth rate and acetic acid formation was comparable to the parent strain in fed-batch. Furthermore it was shown that a higher cell mass was reached using one of the mutants when the cells were cultivated for as long time as possible. The higher cell mass can be used to reach a higher total productivity.</p>