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Item In search of novel folds : protein evolution via non-homologous recombination : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand(Massey University, 2014) Saraswat, MayankThe emergence of proteins from short peptides or subdomains, facilitated by the duplication and fusion of the minigenes encoding them, is believed to have played a role in the origin of life. In this study it was hypothesised that new domains or basic elements of protein structure, may result from nonhomologous recombination of the genes coding for smaller subdomains. The hypothesis was tested by randomly recombining two distantly related (βα)8-barrel proteins: Escherichia coli phosphoribosylanthranilate isomerase (PRAI), and β subunit of voltage dependent K+ channels (Kvβ2) from Rattus norvegicus. The aim was to identify new, folded structures, which may or may not be (βα)8-barrels. Incremental truncation (ITCHY), a method for fragmenting and randomly recombining genes, was used to mimic in vivo non-homologous recombination and to create a library of chimeric variants. Clones from the library were selected for right reading frame and solubility (foldability) of the recombined chimeras, using the pSALect selection system. Out of the six clones identified as soluble by pSALect, only one (P25K86) was found to be actually soluble. The protein, P25K86, was found to form oligomers and on treatment with a reducing agent, β-mercaptoethanol the multimeric state disappeared. The protein has three cysteines and one of the cysteines (Cys56) was found to mediate in the bond formation, thus giving a dimeric state. An engineered version of P25K86 that has the Cys56 replaced by serine was expressed as a monomer and additionally it was found to be ! iv! more stable. As the pSALect folding selection system reported false positives, i.e. only one of the six chimeras was actually soluble, it was concluded that the in vivo solubility selection system was leaky. A series of experiments were conducted so as to improve pSALect that led to the creation of pFoldM – a more stringent selection system, discussed in chapter 4. Comparing the newer improved version with the old, two more interesting chimeras were discovered. A total of 240,000 non-homologous recombination events were created in vitro and three soluble chimeras (evolutionary solutions) were found. Data from circular dichroism spectroscopy (CD) combined with heteronuclear single quantum coherence (HSQC) spectra suggest that the proteins, P24K89 and P25K86, are present in a molten globule state. ITCHY, as a means of mimicking the subdomain assembly model, was applied in vitro. The discovery of two interesting chimeras (P25K86 and P24K89) using highthroughput engineering experiments widens the possibilities of exploring the protein structure space, and perhaps offers close encounters with these never born proteins that may be trapped in an ensemble of fluctuating (structured and unstructured) states.Item Downstream purification and analysis of the recombinant human myelin basic protein produced in the milk of transgenic cows : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University (Palmerston North) New Zealand.(Massey University, 2009) Al-Ghobashy, Medhat Ahmed Abdel-HamidDownstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.