The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.

O cDNA da glicoproteína do vírus da raiva (GPV) foi clonado em vetores plasmidiais (indutíveis) contendo ou não o cDNA do sinal de secreção BiP e da resistência ao antibiótico higromicina B. Esses vetores foram transfectados em células S2 e foram obtidas populações e subpopulações. A população S2MTGPV-H apresentou níveis 5x maiores na expressão da GPV em análise por FACS (~ 50% das células) e por ELISA (~ 0,65 µg/107 células). A seleção de subpopulações permitiu um aumento de aproximadamente 10x na expressão da GPV, especialmente na população S2MTGPV*-H. O tratamento com NaBu resultou em uma redução de aproximadamente 20% no crescimento celular e um aumento de 50% na GPV expressa pela população S2MTGPV*-H (~ 8,3 µg/107 células). O meio de cultura SF900 II permitiu um maior crescimento das células S2MTGPV*-H e uma maior síntese de GPV comparado com outros meios de cultura. Nossos dados mostram que a expressão da GPV pôde ser otimizada através da construção de vetores de expressão/seleção, subpopulações, da exposição da cromatina e do meio de cultura utilizado. / The cDNA encoding the entire rabies virus glycoprotein (RVGP) gene was cloned in plasmids (inductive) with or without a cDNA coding for the secretion signal and coding for the selection hygromicin antibiotic. These vectors were transfected into S2 cells and we had obtain cells populations and subpopulations S2MTRVGP-H cell population were shown to express 5 times higher of RVGP as evaluated by FACS (~ 50 %) and ELISA (~ 0.65 mg/107 cells at day 7). Sub-population selection allowed a higher RVGP expression, especially for the S2MTRVGP*-H. NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MTRVGP*-H (~ 8.3 mg/107 cells at day 7 after induction). SF900II medium leading to a higher S2MTRVGP*-H cell growth allowed a higher final RVGP synthesis in this cell culture. The data show that RVGP synthesis may be optimized by the expression/selection vectors design, cell sub-populations selection, chromatine exposure and culture medium employed.

O cDNA da glicoproteína do vírus da raiva (GPV) foi clonado em vetores plasmidiais (indutíveis) contendo ou não o cDNA do sinal de secreção BiP e da resistência ao antibiótico higromicina B. Esses vetores foram transfectados em células S2 e foram obtidas populações e subpopulações. A população S2MTGPV-H apresentou níveis 5x maiores na expressão da GPV em análise por FACS (~ 50% das células) e por ELISA (~ 0,65 µg/107 células). A seleção de subpopulações permitiu um aumento de aproximadamente 10x na expressão da GPV, especialmente na população S2MTGPV*-H. O tratamento com NaBu resultou em uma redução de aproximadamente 20% no crescimento celular e um aumento de 50% na GPV expressa pela população S2MTGPV*-H (~ 8,3 µg/107 células). O meio de cultura SF900 II permitiu um maior crescimento das células S2MTGPV*-H e uma maior síntese de GPV comparado com outros meios de cultura. Nossos dados mostram que a expressão da GPV pôde ser otimizada através da construção de vetores de expressão/seleção, subpopulações, da exposição da cromatina e do meio de cultura utilizado. / The cDNA encoding the entire rabies virus glycoprotein (RVGP) gene was cloned in plasmids (inductive) with or without a cDNA coding for the secretion signal and coding for the selection hygromicin antibiotic. These vectors were transfected into S2 cells and we had obtain cells populations and subpopulations S2MTRVGP-H cell population were shown to express 5 times higher of RVGP as evaluated by FACS (~ 50 %) and ELISA (~ 0.65 mg/107 cells at day 7). Sub-population selection allowed a higher RVGP expression, especially for the S2MTRVGP*-H. NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MTRVGP*-H (~ 8.3 mg/107 cells at day 7 after induction). SF900II medium leading to a higher S2MTRVGP*-H cell growth allowed a higher final RVGP synthesis in this cell culture. The data show that RVGP synthesis may be optimized by the expression/selection vectors design, cell sub-populations selection, chromatine exposure and culture medium employed.

Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB. / Graduate