<p>The goal of this work was to contribute to the fed-batch process optimisation task by deriving parameters that have considerable impact on productivity as well as product quality The chosen parameters were I) the design of the glucose feed profile, II) the choice of induction strategy, with respect to the method of addition, and III) the time of the induction, with respect to the specific glucose consumption rate. </p><p>The present fed-batch experiments using the lacUV5-promoter, for production of b-galactosidase, have shown that a high glucose feed rate gives a specific production rate, q_p, that is twice as high, after induction, compared to a feed rate that is 2.5 times lower. The constant accumulation of lacZ-mRNA indicates that the translational capacity is initially limiting the synthesis machinery, but after four hours of maximum specific production and a corresponding drop in lacZ-mRNA production, the cultivation is likely to be transcription limited. The high feed-rate system resulted in high accumulation of β-galactosidase, corresponding to 40% of total cellular proteins.</p><p>By design of feed profiles in a fed-batch process the detrimental effects of overflow metabolism, giving acetic acid formation, can be avoided. However, the results show that a one-dose addition of isopropyl-β-D-galactopyranoside (IPTG), provokes a non-growth associated production of acetic acid. This response can be alleviated by; lowering the inducer concentration (in this case to below 165 μM), by further reducing the feed rate of glucose or by using alternative induction methods. The use of a stepwise addition or a feed of IPTG thus delayed and reduced the level of acetic acid accumulation. It was also shown that a small change in the time-point of induction lead to large variability, regarding both productivity and acetic acid accumulation, in a fed-batch cultivation, </p><p>In order to further investigate the protein quality two additional proteins were studied in fed-batch cultivations using high and low glucose feed. The aim was to prove the hypothesis that the feed related change in the rate of synthesis of the nascent polypeptide controls the product quality. For the two proteins: Zb-MalE (wt) and Zb-MalE31 (mutant), the transcription rate, in terms of amount of IPTG, and translation rate, in terms of changes in feed rate, influences the percentage of inclusion body formation and degradation of nascent polypeptide. The data show a higher rate of inclusion body formation for the model protein Zb-MalE31 during high feed rate cultivations, as well as at high levels of inducer. Furthermore, the rate of proteolysis was significantly higher for a high feed rate. The high feed rate thus results in a higher rate of synthesis but a lower corresponding quality, for the model proteins studied.</p><p>In the present investigation of fed-batch cultivations using several different expression vectors, it was found that the central alarmone guanosine tetraphosphate (ppGpp) was formed at both high and low feed rates upon induction. It could be shown, however, that by secretion of Zb-MalE to the periplasm, the stringent response could be avoided. This might be due to the decreased burden on the host where the secretion of product further seems to make the cell able to redirect the carbon flux from overflow metabolism, since no acetic acid was produced. The secretion also demonstrates that the growth arrest could be aborted, which is otherwise gained in the P_malKproduction system.</p><p>A novel fed-batch process based on the promoters for the universal stress proteins A and B (P_uspA, P_uspB) was designed to make use of these powerful promoters in an industrial production context. It was concluded that the process had to start from a high specific growth rate and induction was performed once a limiting feed started. This was done to purposely induce the stringent response and/or acetic acid accumulation since this was required for induction. In the suggested system, induction has to be performed and maintained at continuous substrate feeding, whilst avoiding exceeding the cellular capacity, since the stationary phase starvation alone did not lead to production. In conclusion, a new stress induction based production system was achieved resulting in high accumulations of product protein without any detected metabolic side effects.</p>

Thymidine oligomers were chemically synthesised and linked to available amino functions of transferrin in alternative orientations: (a) A CMP residue attached to the 3' end of (pT)₁₀ with terminal deoxynucleotidyl transferase was oxidised with NaI0 and linked to transferrin via a Schiff base formation. (b) The 5' terminal phosphate group of (pT)₅ was activated with imidazole and reacted with transferrin to form a phosphoramide bond. The (pT)₅ thus attached to the protein was elongated to (pT)₃₀₀ using terminal deoxnucleotidyl transferase and TTP. The latter conjugate was capable of hybridising poly(A) tailed linear PBR322 DNA. The binding of this hybridisation complex to the transferrin receptor on various cell types was investigated. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

Over the past couple of years there has been rapid progress in the development and design of safe and effective delivery systems for the administration of protein and peptide drugs. The effective delivery of these type of drugs are not always as simple as one may think, due to various inherent characteristics of these compounds. Due to the hydrophilic nature and molecular size of peptide and protein drugs, such as recombinant human growth hormone, they are poorly absorbed across mucosal epithelia, both transcellularly and paracellularly. This problem can be overcome by the inclusion of absorption enhancers in peptide and protein drug formulations but this is not necessarily the best method to follow. This investigation focussed specifically on the evaluation of the ability of the PheroidTM carrier system to transport recombinant human growth hormone across mucosal epithelia especially when administered via the nasal cavity. The PheroidTM delivery system is a patented system consisting of a unique submicron emulsion type formulation. The PheroidTM delivery system, based on PheroidTM technology, will for ease of reading be called Pheroid(s) only throughout the rest of this dissertation. The Pheroid carrier system is a unique microcolloidal drug delivery system. A Pheroid is a stable structure within a novel therapeutic system which can be manipulated in terms of morphology, structure, size and function. Pheroids consist mainly of plant and essential fatty acids and can entrap, transport and deliver pharmacologically active compounds and other useful substances to the desired site of action. The specific objectives of this study can be summarised as follows: a literature study on Pheroid technology; a literature study on chitosan and N-trimethyl chitosan chloride; a literature study on recombinant human growth hormone (somatropin); a literature study on nasal drug administration; formulation of a suitable Pheroid carrier; entrapment of somatropin in the Pheroid carrier, and in vivo evaluation of nasal absorption of somatropin in Sprague-Dawley rats. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

The Life Marker Chip instrument, which has been selected to fly as part of the 2018 ExoMars rover mission payload, aims to detect up to 25 organic molecules in martian rocks and regolith, as markers of extant life, extinct life, meteoritic in-fall and spacecraft contamination. Martian samples will be extracted with a solvent and the resulting liquid extracts will be analysed using multiplexed microarray-format immunoassays. The LMC is under development by an international consortium led by the University of Leicester and the work described within this thesis was carried out at Cranfield University as part of the consortium’s broader program of work preparing the LMC instrument for flight in 2018. Within this thesis four specific areas of LMC instrument development are addressed: the investigation of immunoassay compatible liquid extraction solvents, the study of likely interactions of martian sample matrix with immunoassays, the development of antibodies for the detection of markers of extinct life and demonstration of solvent extraction and immunoassay detection in a flight representative format. Cont/d.

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular binding partner of Human Immunodeficiency Virus type 1 (HIV-1) integrase. It is a human nuclear protein, which has been implicated in transcriptional regulation and cell survival. The role of LEDGF/p75 in HIV integration is well characterized, the HIV integrase binding domain (IBD) was identified and structural studies, which provide detail information about this interaction, were done. However, very little is known about its physiological function. As a transcriptional co-activator, LEDGF/p75 is implicated not only in HIV replication, but also in human cancer and autoimmunity. Key feature for both, the viral and cellular role of this protein, is its ability to act as a molecular adaptor tethering proteins to the chromatin fiber. Recently, PogZ (Pogo transposable element derived protein with zinc finger domain) was identified and validated as a new cellular interaction partner of LEDGF/p75. It was shown, that their interaction is mediated by IBD of LEDGF/p75 and the C-terminal domain of PogZ. To gain more insight in this interaction, we have initiated structural studies of their complex. Structural information is crucial for understanding the LEDGF/p75 biological role and might help in design of inhibitors selectively blocking...

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Natural killer cells, or NK cells are an integral component of innate immunity and fullfills the function of recognizing and killing tumor and virus-infected cells. Their function is regulated by signals produced by the interaction of inhibitory and stimulatory receptors on their surface with their specific ligands on the targer cell surface. NKp80 is an activating receptor of NK cells and forms specific complex with cell receptor AICL, both of which belong to the family of C-type lectin-like receptors. Overexpression of AICL receptor is preferably specific for tumor cells of myeloid character. This master's thesis describes the production of AICL mutated form by expression in Escherichia coli BL21 Gold (DE3) followed by isolation and in vitro renaturation of the target protein. In a previous study it was found that an odd number of cysteines in the extracelular lectin domain of AICL causes wrong folding of the protein. Substituting an odd cystein for serine at position 87 lead to stable soluble form of AICL with an even number of cysteines in conserved positions, typical for CTLD receptors. Correctness of the formation of disulfide bonds between cysteines was verified by mass spectrometry. Significant amount of the protein gained allowed for setting up a wide variety of crystallization conditions....

Natural killer or NK cells are immunocytes that mediate innate immunity against pathogens and tumors without pre-exposition to the antigen. They are holding rapid antiviral defense during the initial phase of immune response, before starting the production of antibodies and the development of specific cytotoxic T -lymphocytes. On the surface of NK cells is expressed wide range of inhibition and activation receptors. Important family of those receptors are C - type lectin like from which the family of NKR - P1 ("natural killer cell receptor - protein 1") was discovered first. Diploma thesis deals with the preparation/study of mice NK cell activation receptor NKR- P1C and searching for its binding partner. The soluble form of the protein NKR-P1C was prepared by recombinant expression using the transient transfection of HEK293 cell line (human embryonic kidney 293) with wild type or homogenous glycosylation as IgG - Fc fusion protein, from which was it possible to obtain pure dimer of NKR P1C, after process of affinity purification, TEV protease cleavage and HPLC chromatography. The fusion protein was bound to protein A labeled with a fluorescent probe DyLight 488. Mice tissues and cell lines were labeled by this complex for purpose of seeking ligand.