In proteomic studies, separate experimental protocols have been necessary to identify proteins, determine their function, and predict their three-dimensional structure. In this study, a function-based separation of proteins was conceived to fractionate proteins prior to enzymatic digestion. In the initial demonstration of this technique, a DNA substrate was used to separate the DNA-binding proteins from the rest of the proteins in a lysate in order to identify protein function and to simplify the complex mixture of proteins. A total of 232 putative DNA-binding proteins and over 540 proteins in all were identified from E. coli. Hypothetical or unknown proteins were found, some of which bind to DNA. As a part of this demonstration, changes in protein expression caused by different environmental conditions (aerobic and anaerobic atmospheres) were observed. In a second demonstration, aimed at determining the three-dimensional structure of the DNA binding proteins, binding sites were blocked with oligonucleotides, and the modified proteins were purified, enzymatically digested, and subjected to tandem mass spectrometry. The amino acids in the DNA-binding domains of three proteins were determined. In a final application of function-based separation, DNA-binding proteins were digested with trypsin and the resulting peptides were separated using HPLC and subsequently analyzed using MALDI TOF/TOF and ESI Q-TOF instruments to study the complementary nature of the two ionization techniques, taking into account the differences between the mass analyzers. Based on the analysis of a large data set containing hundreds of peptides and thousands of individual amino acids, some of the currently held notions regarding the ionization processes were confirmed. ESI tends to favor the analysis of hydrophobic amino acids and peptides while MALDI is disposed toward mainly basic and aromatic species. These tendencies in ionization account in large part for the complementary nature of the peptides and proteins identified by the ESI and MALDI instruments and make it necessary to employ both types of instruments to gain the most information out of a given sample in a proteomics study. / Graduation date: 2004

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Bibliography: leaves 215-236. / x, 249 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Scale-up of a biochemical process involving expression of an Insulin-like Growth Factor-I analogue (LongR3IGF-I) as inclusion bodies within the bacterium Escherichia coli has been investigated. The principal focus was directed to the operation of refolding wherein the biological potency of the protein is imparted. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1998?

Bibliography: leaves 215-236. / x, 249 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Scale-up of a biochemical process involving expression of an Insulin-like Growth Factor-I analogue (LongR3IGF-I) as inclusion bodies within the bacterium Escherichia coli has been investigated. The principal focus was directed to the operation of refolding wherein the biological potency of the protein is imparted. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemical Engineering, 1998?

No description available.

Purified macrophage cationic proteins were used in functional assays to determine their interactions with the outer membrane and lipopolysaccharide of Pseudomonas aeruginosa. A fluorescent derivative of polymyxin B (dansyl-polymyxin) was found to bind to saturation to purified lipopolysaocharide, with similar affinity for the aminoglycoside supersensitive strain H215 and wild type strain H103 lipopolysaocharide. MCP-1 could displace more dansyl-polymyxin bound to the lipopolysaocharide of both strains, and bound with greater affinity than MCP-2. When whole cells were used, MCPs also displaced bound dansyl-polymyxin. Effects on the outer membrane of whole cells were examined by determining the initial rate of uptake of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine. Uptake was enhanced in the presence of MCPs, indicating permeabilization of the outer membrane. MCP-1 caused maximal uptake of the probe at 40 µg/ml, MCP-2 at 70 µg/ml, and crude extract at only 20 µg/ml. Uptake of the probe was found to be enhanced at add pH, with maximal uptake occurring with only 7.5 µg/ml MCP-1 at pH 6.5. The data suggested that MCPs act to permeabilize the outer membranes of P. aeruginosa in a manner analagous to that defined for other polycationic agents. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Whole safflower seeds of the Mexican variety Kino'76 with a protein content of 17.30% (dwb) were subjected to the processes of dehulling, defatting (n-hexane extraction) and debittering (70% methanol extraction) to produce four types of meals preparations: whole safflower meal, dehulled safflower meal, debittered, whole meal and debittered, dehulled meal with protein contents of 26.90, 66.93, 26.70 and 69.92%, respectively. The proteins of each meal were studied in detail by means of protein fractionation, gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Osborne solubility fractionation of the protein of whole safflower meal showed that the amount of protein in the alkali soluble fraction was approximately 71% of the total and the alcohol soluble fraction did not contain any protein. After dehulling and debittering, the amount of protein in the alkali soluble fraction decreased by 30%, whereas the amount of protein in the insoluble residue increased by 12%. SDS-PAGE of the proteins of the water-, salt- and alkali soluble fractions revealed that they consisted of 8, 13 and 13 distinct subunits, respectively, with apparent molecular weights ranging from 14.7 to 88.0 kDa. The number of subunits and molecular weight distribution decreased as a result of debittering. Fractionation of the proteins of each meal by gel filtration chromatography followed by SDS-PAGE demonstrated that proteins of safflower seed are highly heterogeneous. The process of debittering caused major alteration of the molecular weight profile and subunit composition of the gel filtration protein fractions.