Effective methods for isolation and purification of glycoproteins are of increasing significance to the rapidly growing biopharmaceutical and diagnostic industry. Glycoproteins represent the majority of therapeutic proteins on the market and are effectively used to treat immune disorders, infections, cancers, and other diseases. Targeting these glycoproteins is also critical to an emerging field of glycoproteomics aimed to understand structure-function relationships of glycans. Architecturally, these glycoproteins are proteins with covalently linked oligosaccharide chains of varying monosaccharide composition. Affinity chromatography has proven to be an excellent method of glycoprotein purification at the bench scale. However, chromatography in large scale production has its drawbacks. Column fowling, flowrate limitations, and diffusional constraints collectively hinder the effectiveness of the method. An alternative proposed in this dissertation is the use of affinity precipitation as a purification technique. The three main objectives are 1) develop and produce dual-functional, thermo-responsive affinity ligands from a biological host, 2) characterize and optimize the accompanying affinity precipitation method, and 3) apply the ligand and process to relevant, unmodified glycoproteins. The design of the thermo-responsive affinity construct was comprised of two main functional domains. The binding capability was achieved by selection of small ligands with affinity to a specific monosaccharide moiety. Two different lectins, or sugar binding proteins, were used in the fusion design: a fucose binding lectin from Ralstonia solanacearum, and a sialic acid binding lectin from Vibrio cholera. The thermo-responsive functionality was obtained by use of an elastin-like peptide (ELP), which confers inverse solubility relationship properties to the fusion construct. A small library of varying ELP chain lengths were designed to find the optimal size fusion for both production and function. These dual functional ligands were cloned and expressed in the microbial host, E. coli. Furthermore, secretion of these constructs was achieved by employing the Tat secretion pathway in combination with an outer membrane lipoprotein deletion mutant with a leaky periplasm phenotype. This secretory mechanism allows for easy isolation, avoidance of inclusion bodies, and no additional protease inhibitors. After successful production, the ligands were tested to confirm that dual functionality was preserved in fusion form. Once binding conditions and precipitation properties were ascertained, the purification ability was tested on model glycoproteins. Experimentation was carried out monitoring the purification yield, purity, and retained activity of the target enzymes. High contaminant solutions, such as cell lysates, were spiked with the model glycoproteins to mimic crude protein solutions. The purification ability of the constructs in these models was observed. The method was then implemented on two relevant glycoprotein applications: 1) purification of soybean peroxidase from a crude protein extract and 2) targeting the therapeutic protein erythropoietin from albumin rich, used CHO cell media. By implementation of the fucose targeting fusion construct, the unmodified soybean peroxidase is isolated from a natural crude extract from the soybean hull, a by-product of the soybean industry. The affinity precipitation method parameters were optimized with respect to ratios, temperatures, recycle, and elution buffers to achieve successful isolation of the low abundance enzyme. Under the optimized conditions, >95% recovery yield and a purification of 22.7 fold of an active, pure product was attainable. The purification of erythropoietin led to additional experimentation with high-abundant glycoprotein solutions, as well as expansion of the affinity ligand platform. The concept of multi-lectin affinity precipitation, using the fucose and sialic acid binding lection sequentially, was introduced and tested for purification capability. An industrially relevant scheme involving isolation of the erythropoietin from used CHO cell media allowed for an achievable yield of about 60%, with a resulting albumin depletion of about 85%. In addition to development of a pair of novel thermo-responsive affinity ligands for glycoprotein purification, this dissertation provides insight on possible improvements and future directions with respect to the thermo-responsive affinity ligand platform. This unique concept employs novel lectin fusions to target valuable glycoproteins using a method avoiding the major drawbacks associated with chromatography.

O gene do Retardo Mental do X Frágil (FMR1) possui 17 éxons e seu transcrito primário pode sofrer splicing alternativo, havendo, entre outros eventos, possibilidade de exclusão ou inclusão do éxon 12. O produto da expressão do FMR1, a proteína do retardo mental do X frágil (FMRP), possui papéis importantes no sistema nervoso central, atuando como repressora da tradução de RNAm em espinhas dendríticas e controlando a síntese de proteínas envolvidas na função sináptica. Entre dois domínios centrais do tipo KH presentes na FMRP, o segundo (KH-2) é responsável pela interação da proteína aos polissomos. O domínio KH-2 é codificado pelos éxons 9 a 13 do FMR1 e possui a alça variável mais longa já observada entre proteínas humanas, que é codificada pelos éxons 11 e 12. A inclusão do éxon 12 no RNAm do FMR1 causa uma extensão em fase dessa alça variável do KH-2 da FMRP. Estas isoformas apresentam expressão significativa em neurônios cortico-cerebrais e cerebelares do rato, no primeiro mês pós-natal. Este trabalho baseia-se em resultados prévios do grupo de pesquisa, em que se identificaram sequências curtas no íntron 12 do FMR1, com potencial para agir como acentuadores de splicing. Baseando-nos na hipótese de que essas sequências constituem elementos transcritos que se ligam a fatores proteicos do núcleo celular, potencialmente reguladores do splicing do pré-RNAm do FMR1, realizamos ensaios de precipitação por afinidade com extratos nucleares de córtex cerebral de rato e transcritos do loco, biotinilados. Análises por espectrometria de massas revelaram enriquecimento de proteínas nucleares, contendo domínios de ligação a RNA, principalmente aquelas relacionadas à regulação e processamento de pré-RNAm, sobretudo o splicing / Fragile X Mental Retardation 1 gene (FMR1) comprises 17 exons. Its primary transcript is subject to alternative splicing, allowing for the possibility of exon 12 inclusion or skipping, among other events. The product of FMR1 gene expression, fragile X mental retardation protein (FMRP), has important roles in the central nervous system, acting as a translational repressor in dendritic spines, thus controlling the synthesis of proteins involved in synaptic function. FMRP has two central KH domains. One of them (KH-2) is responsible for its interaction with polysomes. The KH-2 domain is encoded by FMR1 exons 9 to 13. It contains the longest variable loop ever observed among human KH-containing proteins, which is encoded by FMR1 exons 11 and 12. Exon-12 inclusion in FMR1 mRNA causes an in-frame extension of FMRP KH-2 domain variable loop. These isoforms appear significantly expressed in cortico-cerebral and cerebellar neurons of the rat in the first month after birth. We have previously identified short sequences within FMR1 intron 12 that may potentially act as splicing enhancers. Our study is based on the hypothesis that those sequences when transcribed should bind to nuclear protein factors that may function as FMR1 exon 12 pre-mRNA splicing regulators. To initiate an experimental approach to test that hypothesis, we conducted affinity precipitation assays with rat cerebral cortex nuclear extracts and biotinylated transcripts. Mass spectrometry analyses disclosed proteins that have been described to be enriched in the cell nucleus, contain RNA-binding domains, and be functionally related to pre-mRNA processing, notably splicing

O gene do Retardo Mental do X Frágil (FMR1) possui 17 éxons e seu transcrito primário pode sofrer splicing alternativo, havendo, entre outros eventos, possibilidade de exclusão ou inclusão do éxon 12. O produto da expressão do FMR1, a proteína do retardo mental do X frágil (FMRP), possui papéis importantes no sistema nervoso central, atuando como repressora da tradução de RNAm em espinhas dendríticas e controlando a síntese de proteínas envolvidas na função sináptica. Entre dois domínios centrais do tipo KH presentes na FMRP, o segundo (KH-2) é responsável pela interação da proteína aos polissomos. O domínio KH-2 é codificado pelos éxons 9 a 13 do FMR1 e possui a alça variável mais longa já observada entre proteínas humanas, que é codificada pelos éxons 11 e 12. A inclusão do éxon 12 no RNAm do FMR1 causa uma extensão em fase dessa alça variável do KH-2 da FMRP. Estas isoformas apresentam expressão significativa em neurônios cortico-cerebrais e cerebelares do rato, no primeiro mês pós-natal. Este trabalho baseia-se em resultados prévios do grupo de pesquisa, em que se identificaram sequências curtas no íntron 12 do FMR1, com potencial para agir como acentuadores de splicing. Baseando-nos na hipótese de que essas sequências constituem elementos transcritos que se ligam a fatores proteicos do núcleo celular, potencialmente reguladores do splicing do pré-RNAm do FMR1, realizamos ensaios de precipitação por afinidade com extratos nucleares de córtex cerebral de rato e transcritos do loco, biotinilados. Análises por espectrometria de massas revelaram enriquecimento de proteínas nucleares, contendo domínios de ligação a RNA, principalmente aquelas relacionadas à regulação e processamento de pré-RNAm, sobretudo o splicing / Fragile X Mental Retardation 1 gene (FMR1) comprises 17 exons. Its primary transcript is subject to alternative splicing, allowing for the possibility of exon 12 inclusion or skipping, among other events. The product of FMR1 gene expression, fragile X mental retardation protein (FMRP), has important roles in the central nervous system, acting as a translational repressor in dendritic spines, thus controlling the synthesis of proteins involved in synaptic function. FMRP has two central KH domains. One of them (KH-2) is responsible for its interaction with polysomes. The KH-2 domain is encoded by FMR1 exons 9 to 13. It contains the longest variable loop ever observed among human KH-containing proteins, which is encoded by FMR1 exons 11 and 12. Exon-12 inclusion in FMR1 mRNA causes an in-frame extension of FMRP KH-2 domain variable loop. These isoforms appear significantly expressed in cortico-cerebral and cerebellar neurons of the rat in the first month after birth. We have previously identified short sequences within FMR1 intron 12 that may potentially act as splicing enhancers. Our study is based on the hypothesis that those sequences when transcribed should bind to nuclear protein factors that may function as FMR1 exon 12 pre-mRNA splicing regulators. To initiate an experimental approach to test that hypothesis, we conducted affinity precipitation assays with rat cerebral cortex nuclear extracts and biotinylated transcripts. Mass spectrometry analyses disclosed proteins that have been described to be enriched in the cell nucleus, contain RNA-binding domains, and be functionally related to pre-mRNA processing, notably splicing