Made available in DSpace on 2014-10-09T12:55:16Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:06:00Z (GMT). No. of bitstreams: 0 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A Prolactina humana hPRL é um hormônio protéico com 199 aminoácidos (MM ~ 23.000 Da) com um amplo espectro de atividades biológicas, sendo mais conhecido por estimular a lactação e regular o crescimento e diferenciação da glândula mamária. Além de quebra proteolítica, a maioria dos variantes de prolactina podem ser resultantes de outros processos pós-traducionais como polimerização, fosforilação, desamidação, sulfatação e glicosilação. Essa proteína contém apenas um sítio potencial de glicosilação por ligação à asparagina, localizada no aminoácido 31, que é parcialmente ocupado (10%) quando a proteína é sintetizada em células eucariotas. Apesar da atividade biológica in vitro da prolactina glicosilada (G-hPRL) ser muito menor (~4 vezes) quando comparada à não glicosilada, sua função fisiológica ainda não é bem definida e a porção de carboidrato parece ter um importante papel na biossíntese, secreção, atividade biológica, e clearance plasmático do hormônio. Com o objetivo de melhor caracterizar e estudar esta variante hormonal, foi realizada sua purificação em escala laboratorial a partir de células de ovário de hamster chinês (CHO) modificadas geneticamente, utilizando meio de cultura suplementado com cicloheximida, aumentando ~4 vezes sua concentração absoluta e ~10 vezes a razão entre a isoforma glicosilada e a não-glicosilada. A purificação da G-hPRL seguiu um processo simples e efetivo de duas etapas principais baseado em uma coluna de troca catiônica e uma coluna preparativa de exclusão molecular acoplada a um sistema de cromatografia líquida de excusão molecular de alta eficiência (HPSEC). A caracterização foi feita por HPLC de fase reversa (RP-HPLC) e exclusão molecular, SDS-PAGE, Western Blotting, espectometria de massa (MALDI-TOF) e um bioensaio in vitro utilizando células Nb2 e Ba/F3-LLP. Nossos resultados demostram que a cicloheximida pode ser uma importante ferramenta para aumentar a produção de prolactina glicosilada, facilitando a purificação e caracterização dessa isoforma. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP / FAPESP:06/52973-9

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils.

Recombinant protein technologies have emerged as important tools for the production of proteins with industrial, academic and biopharmaceutical applications. However, current process development for a target protein is hindered by the burden recombinant protein expression places on the host system, with the level of this negative effect and the ideal production conditions varying from protein to protein. As a result current process optimisation relies on trial and error to determine the optimal set up for a given target protein. The work presented here will examine two mechanisms by which this burden acts on the cell, contributing towards making the overall burden effect and the optimal process conditions more predictable. Flux Balance Analysis was used to examine the effect of amino acid supplementation on the metabolic cost of a recombinant protein to predict which supplements would improve the efficiency of production, predictions supported elsewhere in the literature. However, experimental validation in batch and fed batch cultures for the production of human Granulocyte Colony Stimulating Factor demonstrated that these supplementation strategies do not lead to an increase in yield or performance. The results from the computational modelling alongside similar studies in the literature suggest that the more important factor may be optimisation for better growth generally rather than targeted attempts based on the protein composition. The sensitivity of native E. coli proteins to a loss of chaperone activity was predicted using the solubility data of the eSol database, identifying rrmJ as a protein of interest. The possible significance of rrmJ for chaperone saturation was examined alongside examining the effect of recombinant protein solubility using Green Fluorescent Protein (GFP) and its mutant GFP_A which have differing solubility. However, neither an effect through rrmJ nor a negative effect of recombinant protein solubility on growth was identified. Kinetic modelling for a mechanistic examination of the chaperone network suggests this is because the poorly folding protein is preferentially shifted to the insoluble fraction while the better performing proteins, i.e. the native proteins, are relatively unperturbed despite saturation of the chaperones. Overall the study was not able to make these areas more predictable. However, the observations made within this study contribute to an improvement in our understanding of two key mechanisms of interest in the field. Of particular interest is the identification of two logical hypotheses within the literature to be, at least in the cases tested, false.

As the worldwide demand for recombinant proteins and valuable metabolites continues to grow, and as the biological toolset at our disposal continues to expand, the development of novel, robust, and effective platforms for the production of these bioproducts represents an area of ever-increasing interest. Although many such bioprocesses are currently economically viable, many more, though holding considerable promise, remain uncompetitive. The development of novel, more productive systems increases the versatility and industrial applications of bioprocesses. The work described in this thesis explores several aspects of bioprocessing, both on the upstream side, concerned with the development of novel recombinant protein expression platforms or the isolation of novel genes with products possessing characteristics of interest, and on the downstream side, through the improvement of fermentation-based bioprocesses. Thirty-six homoplasmic recombinant strains of the microalga Chlamydomonas reinhardtii were developed having integrated genes for phytase or xylanase under the control of psbA and psbD promoters, codon optimized using novel algorithms, at two different genetic loci, in chloroplasts, to be used as novel animal feed additives. Enzyme production was characterized, and results, when compared to other published work in this field, may provide insight into the factors impacting recombinant protein production in microalgae. Using a “bio-prospecting approach”, the microflora of the digestive tract of a Canadian beaver was screened for cellulase-producing microorganisms. Although the screening approach did successfully identify a novel β-glucosidase gene from an isolated strain of Bacillus thuringiensis, the sequence was not significantly different from those already characterized. Two bioprocessing studies were performed to improve recombinant protein production in Pichia pastoris. In the first, the composition of standard Basal Salt Medium (BSM) was systematically optimized for the production of recombinant phytase, and the optimized media produced significantly more enzyme than the standard one, while also containing significantly reduced concentrations of KH2PO4 and MgSO4·7H2O (27.9 g/l and 4.8 g/l respectively), lowering the price of process inputs. The second was based on the screening of unconventional carbon sources for candidates that could sustain the growth and enzyme production using the same P. pastoris strain. Fructose and ethanol have shown to be viable alternatives to glucose or glycerol as sole carbon sources, and provide flexibility in terms of process design.

Two separate systems were developed for the expression of recombinant metalloproteins. A synthetic gene encoding the lipase-solubilized form of bovine liver microsomal cytochrome b₅ was designed and assembled for expression in E. coli. Analysis of the initial recombinant cytochrome revealed differences in several physical characteristics of the molecule compared to the authentic bovine liver species, including a reduction potential that was lower by 17 mV. Further studies showed the primary sequence of the initial recombinant differed from the authentic protein in the amidation status of three residues which, when corrected yielded a recombinant protein identical in behaviour to the authentic protein. The participation of Ser64 in the stabilization of the oxidized form of cytochrome b₅ was investigated using site-directed mutagenesis to alter this residue to Ala, which was predicted to ablate a hydrogen bond formed between the protein and heme-propionate 7. Spectroelectrochemical analysis of this variant showed that the reduction potential had been shifted downwards by 7 mV, in contrast to predictions from a structural model describing the red/ox behaviour of cytochrome b₅ (Argos and Mathews, 1975). The role of heme carboxylates in determining the reduction potential was confirmed for both the wild-type and Ala64 variants by heme replacement studies using the esterified derivative of protoporphyrin IX, suggesting that the presence of free carboxylates contributes to the stabilization of the oxidized species. In addition, constructions for the expression of the trypsin-solubilized form of bovine liver microsomal cytochrome b₅ and the erythrocytic form of human cytochrome b₅ are described. A tissue culture cell system was developed for the expression of the N-terminal half molecule of human serum transferrin. The recombinant molecule (hTF/2N) was secreted at high levels from selected eukaryotic cells, and displayed high identity with the proteolytically-derived molecule from authentic human serum transferrin as judged by sequence analysis, electrophoretic mobility and iron binding capacity. A construction for the expression of the C-terminal half molecule was assembled but failed to express recombinant protein when introduced into tissue culture cells. The production of these two heterologous expression systems allows for high-level recovery of recombinant protein and provides a convenient approach to structure-function studies employing site-directed mutagenesis techniques. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Specific Drosphila tRNA Ser4,7 plasmids were identified and isolated by hybridization with purified [¹²⁵I] tRNA Ser4,7 molecules. Seven clones were isolated carrying the Drosphila Ser tRNA Ser4,7 gene and were further characterized by restriction endonuclease digestion; agarose gel electrophoresis and hybridization with individual purified [¹²⁵I] tRNA Ser4,7 molecules. The results show that five different DNA fragments have been isolated, four which code for a single, specific isoacceptor, and one which appears to code for two different isoacceptors. Two plasmids which initially contained multiple Hind III inserts upon primary isolation were recloned to contain single Hind III inserts containing the tRNA Ser4,7 gene. One of these recloned plasmids contained a smaller tRNA Ser4,7 gene carrying insert than did its original multiple insert isolate. Small tRNA Ser4,7 gene carrying restriction fragments were labelled with T4 polynucleotide kinase and [³²P] ATP, strand separated, and electroeluted, in preparation for nucleotide sequencing. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (MSc. (Microbiology)) -- University of Limpopo, 2015 / S. cerevisiae has many properties which have made it the preferred host for the expression and production of a number of recombinant proteins. Xylose is the second most abundant sugar in nature and S. cerevisiae has been engineered to grow on this abundant sugar. Therefore, identifying S. cerevisiae promoters that are strongly induced during growth on xylose will be important in the production of recombinant proteins for the biofuel and other industries. Since xylose is not a native substrate for S. cerevisiae, it is not known how S. cerevisiae promoters will react during growth on xylose. The objective of the study was to evaluate the expression of a reporter gene, the Trichoderma reesei xylanase 2 (XYN2), under the control of five commonly used expression promoters (GPD3, ENO2, PGK1, ADH2 and YG100). Five episomal expression vectors were constructed for this purpose. These vectors were transformed to a recombinant xylose utilizing S. cerevisiae. Xylanase activity assays were used to determine the expression level from each of these promoters. The PGK1 promoter was observed to be the strongest promoter with average activity/OD of 130 nkat/ml/OD on both xylose and glucose. The GPD3 promoter showed the highest average activity/OD of 150 nkat/ml, but xylanase was only produced during growth on glucose. The data presented show that xylose is not a better carbon source than glucose for recombinant protein production in terms of the S. cerevisie promoters evaluated. Further research is required to obtain a yeast strain that grows well on xylose and promoters that show higher level on protein production. Keywords: xylose, promoter, expression, recombinant, S. cerevisiae

Homeologous recombination refers to genetic exchanges between DNA partners containing similar but not identical DNA sequences. Heteroduplex intermediates in such exchanges are expected to contain multiple DNA mismatches at positions of sequence divergence and hence are potential targets for mismatch correction. Thus recombination of this type is of particular interest in understanding the role of DNA mismatch correction on recombination fidelity. Previous studies that examined this question in prokaryotic systems suggested that mismatch repair acts as a barrier to recombination between diverged sequences. The central hypothesis of this thesis is that mismatch correction acts as a barrier to homeologous recombination in yeast. The objectives of these studies was to elucidate the role of mismatch correction in homeologous recombination as a means of dissecting its mechanism in eukaryotic organisms. To examine homeologous genetic events in yeast, I developed an in vivo assay system to evaluate recombination between diverged DNA sequences. A homeologous gene pair consisting of Saccharomyces cerevisiae SPT15 and its Schizosaccharomyces pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene. Each gene carried a different inactivating mutation, rendering this starting strain Spt15-. Recombinants that regenerated SPT15function were identified by genetic selection and the rates of recombination in different backgrounds were compared. The homeologous mitotic recombination assay was utilized to test the role of S. cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. In strains wild type for mismatch repair, homeologous recombination was reduced 150-180 fold relative to homologous controls, indicating that multiply mispaired sequences act in cis as part of an inhibitory mechanism. In the upstream orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17-fold and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1deletion mutant did not exhibit elevated homeologous recombination in either case. Next, I investigated whether mismatch correction acts as a specific or general obstacle to homeologous recombination by blocking one or many exchange pathways. To answer this question, I performed structural analysis on numerous recombinant products from each strain to determine the percentage of products that fell into a given class (crossovers or gene conversions). Each percentage was then multiplied by the overall rate to arrive at a rate of recombination for individual events. Typically 90-100% of homologous and homeologous recombinant products could be accounted for, either as crossovers or gene conversions. Recombination for all classes of products was inhibited when divergent sequences were present, indicating that homeology blocks formation of both crossovers and gene conversions. Sequence analysis of a limited number of homeologous recombinants indicated that transfer of DNA occurred in continuous stretches and that endpoints fell within regions of 3-11 base pairs of perfect homology. Mutations in the mismatch repair genes MSH2 or MSH3that elevate the overall rate of homeologous recombination produced similar rate increases in formation of each recombinant class. This suggests that mismatch correction proteins block multiple types of homeologous recombination events. Taken together, these results support the hypothesis that homeologous and homologous recombination occur by the same (or similar) pathways, with mismatch repair superimposed as an extra level of control over the fidelity of the process. I also investigated whether this homeologous recombination system would be useful in a genetic screen to identify novel genes or new alleles of genes known to increase exchanges between diverged DNA sequences. Hyperhomeologous recombination mutants were isolated following ethylmethane sulfonate mutagenesis of yeast that harbored the spt15 homeologous duplication. Preliminary characterization of these mutants demonstrated that some of these isolates yielded phenotypes that were consistent with mutations in mismatch repair genes verifying the utility of this method to identify such mutants. To improve the use of this system for a mutant screen, I developed a second generation homeologous duplication using URA3. These new starter strains are expected to be important for efficient isolation and characterization of hyperhomeologous recombination mutants.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1980 / Vita. / Includes bibliographical references. / by James F. Gusella. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

To compare the efficacy and duration of the immune response to local and systemic vaccination, Balb/c mice were vaccinated either intraperitoneally (i.p.) with an inactivated A/PR/8/34 (H1N1) vaccine or intranasally (i.n.) with a vaccinia recombinant containing the H1 gene of influenza. The i.p. inactivated vaccine stimulated high serum IgG anti-influenza titres and protected the lungs against viral challenge for the duration of the experiment (17 months). Little nasal wash IgA was induced and the noses were susceptible to challenge. Animals vaccinated i.n. with the recombinant had lower serum IgG titres and the lungs showed poor protection against challenge. Nasal wash IgA titres were higher, however, and the noses were largely protected from viral challenge for 17 months.