Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

<p>Mantle cell lymphoma (MCL) is one of several sub-types of B-cell lymphomas. The malignancy is very aggressive and average survival time is short. The hallmark of MCL is over expression of cyclin D1, however about 15% of all MCL cases do not display this over expression and are easily misdiagnosed. Recently the transcription factor Sox11 has been shown to be specifically over expressed in the nucleus of MCL-tumour cells, and polyclonal rabbit anti-Sox11 antibodies have been used to successfully identify MCL in both cyclin D1 positive and negative cases. Howev-er, human recombinant MCL-specific antibodies as have several advantages over these polyclonal rabbit antibodies; they can easily be produced in large quantities in vitro, their specificity is constant from batch to batch and they can possibly be used for therapeutic purposes. Because of this, it is desirable to produce human recombinant antibodies against proteins over expressed in MCL. In this study human recombinant IgGs have been produced towards two pro-teins over expressed in MCL, Sox11 and KIAA0882. This was done by cloning of single chain variable fragments (scFvs), previously selected from a large scFv library through phage display selection against Sox11- and KIAA0882-protein epitope signature tag (PrEST), into vectors containing human IgG constant regions followed by expression of human IgG antibodies in human embryonic kidney (HEK) 293 cells. One IgG clone for each antigen was shown to be functional and specific. Both clones were shown to have overlapping binding epitopes with their polyclonal rabbit antibody counterpart (rabbit anti-Sox11/KIAA0882) through competitive ELISA. The anti-Sox11 IgG was able to detect two bands in cell lysate in Western blot, of which one probably is Sox11 while the other band possibly could be Sox4. However, this needs to be confirmed in future experiments. The affinity of the anti-Sox11 IgG was measured in Biacore and compared to the affinity of its original scFv. This gave a rough estimation of the affinities, but the values are unreliable and the measurements need to be redone. Although more work has to be put into evaluating the potential of the produced IgGs, they compose a promising starting point to an improved understanding and improved diagnosis of MCL.</p>

Post-weaning diarrhea is a major health challenge in the swine industry and is routinely managed by fortifying pig starter diets with antimicrobials. But there are concerns about antibiotic resistance, hence the need for identifying effective alternatives. The use of spray-dried whole egg powder containing anti-F4 antibodies (SDWE) against recombinant F4 antigens and chitosan oligosaccharide and Enterococcus fecalis probiotic combination (CPRO) was investigated in two trials using enterotoxigenic Escherichia coli K88+ (ETEC) oral challenge model in 21-d-old piglets. Pre-challenge, SDWE supported higher (P < 0.05) piglet performance whereas during the post-challenge period, SDWE and CPRO had no effect on growth performance but diarrhea incidences and severity were reduced (P > 0.05) in SDWE-fed piglets compared to the control. The results show that SDWE supported greater piglet performance pre-ETEC challenge although there was no benefit of SDWE or CPRO supplementation evident during the post-challenge period in early-weaned pigs. / October 2015

The effects of insulin on milk production were tested employing two different approaches. Firstly, 12 Holstein cows were used to determine the effects of feeding calcium propionate (Ca prop) on dry matter intake (DMI) and production traits. The experimental design was a switchback with 2 treatments (Ca prop at 0 or 300 g/d). The DMI was lower when animals received Ca prop. Ca prop did not affect the yield of milk and its components; however, Ca prop increased protein content. The (acetate+butyrate)/propionate ratio in rumen fluid 2 h after feeding was lower when cows received Ca prop. Plasma insulin concentration was not different between treatments and the putative effect of propionate as an insulin secretagogue was probably related to the maintenance of insulin levels when DMI was lower. In conclusion, Ca prop is a potential feed ingredient to increase protein content in milk. The second approach consisted of intravenous infusion of insulin. A trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST) and their interaction in lactating dairy cows. Eight Holstein cows were used in a Latin Square design with 4 treatments: (1) intravenous infusion of saline, (2) infusion of saline and administration of 40 mg of rbST per day, (3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion and (4) rbST administration combined with insulin and glucose infusion. The theory that rbST causes a peripheral resistance to insulin was confirmed. Insulin infusion increased percent protein, percent casein and decreased milk urea content regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent and lactose yield, there was an interaction between insulin and rbST administration. Similarly, there was an interaction between insulin and rbST on plasma IGF-I levels. Fat yield was higher, with a higher content of long chain fatty acids, during rbST administration, regardless of insulin infusion. I

Thesis (D.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 xi, 209 leaves / Protein engineering technologies, such as directed evolution and DNA recombination, are often used to modify enzymes on a genetic level for the creation of useful industrial catalysts. Pre-treatment of paper pulps with xylanases have been shown to decrease the amounts of toxic chlorine dioxide used to bleach pulp. This study was undertaken to improve the thermal and alkaline stabilities of the xylanase from the fungus Thermomyces lanuginosus using ep-PCR and DNA shuffling.

A platform for vaccine development has been developed at Georgia State University utilizing recombinant Salmonella typhimurium flagellin (FliC) fused to an antigen that can be overexpressed in Escherichia coli grown in a two-stage fermentation. The flagellin acts as an adjuvant to increase the immunopotency of the fused antigen. Flagellin is the ligand for Toll-like Receptor 5 (TLR5), a part of the innate immune system. Binding of the flagellin:antigen recombinant protein to TLR5 triggers a strong innate and adaptive immune response to the fused antigen leading to a potentially strong protective immunity to the antigen. Purification of the recombinant FliC fusion protein must meet rigorous criteria in order to be used as a vaccine. One of the major issues in purifying recombinant proteins expressed in a Gram-negative bacterium is the removal of endotoxin. Small amounts of endotoxin present in a vaccine can lead to serious complications, including death. Recombinant proteins are also expressed as either soluble or insoluble protein when over expressed in E. coli. Soluble proteins expressed by the bacterium are properly folded and biologically active, however removal of contaminants such as endotoxin, can be problematic. Insoluble protein is improperly folded and biologically inactive. The insoluble proteins aggregate into inclusion bodies with little or no contaminants associated with the protein, making purification easier. However, in order to restore the biological activity of the insoluble protein, it must first be solubilized and then refolded. This process is often expensive and time consuming, as there is currently no standardized method for protein refolding. In this study a purification method for the soluble protein of two FliC constructs, full-length FliC and FliC fused to a Marburg virus antigen, was evaluated for effectiveness in purification, removal of endotoxin and maintaining TLR5 activity. The proteins of interest were purified utilizing only the soluble protein containing the properly folded and biologically active recombinant protein. Utilizing methods for purification that take advantage of physical and chemical properties of the protein the recombinant proteins were purified and the level of endotoxin reduced to levels acceptable for use as a vaccine. The TLR5 activity of the soluble recombinant proteins was compared to recombinant protein that had been purified using a denaturing and refolding step. The soluble protein elicited a higher TLR5 response at a lower concentration of protein than the refolded protein. Purification of the soluble fraction also involved fewer step and less time than purification of both the soluble and insoluble protein.

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

Disposable technology has gained increasing acceptance in the biopharmaceutical industry over the last decade, and provides many advantages over conventional stainless steel equipment. Disposable rocking bioreactors (RBs) are widely employed for cultivation of recombinant mammalian and insect cell lines, although the perception of inadequate mass transfer has prevented their application to bioprocesses based on microbial platforms. In an effort to thoroughly evaluate the suitability of disposable RBs for cultivation of aerobic microorganisms, a comparative study of one-dimensional (1D) and two-dimensional (2D) disposable RBs, and the conventional stirred tank reactor (STR) was performed. The comparison involved: 1) physical characterization of oxygen mass transfer efficiency and mixing intensity, 2) batch cultivation of Escherichia coli BL21 for comparison of growth characteristics, and 3) batch cultivation of recombinant E. coli BL21 expressing a clinical therapeutic, hCD83ext (the extracytoplasmic domain of human CD83). Oxygen mass transfer (evaluated as the mass transfer coefficient, kLa) was comparable between the 1D RB and STR (approximately 150 h-1) at low working volume (WV), declining linearly with increasing WV, while kLa was highest in the 2D RB for all tested WVs, providing the maximum kLa (394 h-1) at 3 L WV. Fast mixing (t95 of 8-20 s) was observed in all three systems for water and aqueous carboxymethylcellulose (CMC) solutions. Batch growth characteristics of E. coli BL21 were similar in each system, although acetate accumulation was significant in the 1D RB. Batch production of GST-hCD83ext (glutathione S-transferase-hCD83ext fusion protein) resulted in similar soluble protein yields and inclusion body formation between bioreactors. Although cell growth and protein expression were comparable between all bioreactors, the 1D RB is not considered a suitable cultivation system for E. coli under experimental conditions given the significant acetate accumulation observed and high supplemental oxygen requirement for low cell density cultures. On the other hand, considering its formidable mass transfer capacity and overall performance in batch cultivations, the CELL-tainer® is an attractive alternative to the STR for cultivation of recombinant E. coli expressing high value therapeutic proteins.

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.