We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&amp / #945 / A plasmid and the constructed system within the plasmid, pPICZ&amp / #945 / A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations. Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm / N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density.

The purpose of the study is to purify human growth hormone from the fermentation broth by affinity chromatography. For this purpose, human growth hormone specific oligonucleotide aptamers are selected among an aptamer library / selected oligonucleotides were synthesized and used as ligands. Effect of pH on ligand-human growth hormone complex formation was investigated and the highest complex formation was obtained at pH= 7.0. Human growth hormone is separated from the fermentation broth with 99.8% purity and 41% overall yield. The equilibrium data obtained was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are calculated as 0.338 mg hGH/&igrave / mol aptamer and 0.059 mg hGH/ml, respectively. Further, equilibriumdata obtained using aptamer affinity column was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are 0.027 mg hGH/&igrave / mol aptamer and 1.543 mg hGH/ml, respectively. It is possible that, selected aptamer can be used for purification of bulk amounts of recombinant human growth hormone by using aptamer affinity chromatography.

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations / expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.

In this study, the aim was to achieve high concentration of recombinant human growth hormone (rhGH) production by recombinant Pichia pastoris by investigating the effects of various operation parameters and to determine the suitable peptide ligand sequence that shows affinity and specificity to hGH. In this context, firstly the effect of temperature and Tween-20/80 addition on production and cell growth were investigated. While at T=30 and 32&deg / C, there was no difference, at 27 and 25&deg / C cell growth slowed down and production decreased significantly. The addition of Tween-20/80 in existence of co-substrate sorbitol did not affect the bioprocess while in absence of sorbitol Tween alone did not show the same positive effect on product formation and cell growth. Thereafter at T=30&deg / C, without addition of Tween, three sets of pilot scale bioreactor experiments were performed. In the first set, the effect of methanol feeding rate on bioprocess characteristics were investigated at the specific growth rates of &mu / =0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at &mu / =0.04 h-1, the highest rhGH concentration was achieved at &mu / =0.03 h-1. Secondly, conducting methanol feeding at &mu / =0.03 h-1, pH=5.5 experiment was conducted. The highest cell concentration, 45 g L-1 and maximum rhGH concentration 0.25 g L-1 were achieved at t=18 h of the process. Finally, the effect of batch sorbitol feeding on bioprocess was observed by the addition of 50 g L-1 sorbitol at t=0, 14 and 31 h of the production phase. It was shown that sorbitol addition to the medium increased process duration / hence cells enter stationary phase after a longer production phase. However, the protease concentration continued increasing with respect to time and at the end of the process reached twice the concentration it was obtained with single sorbitol addition case decreasing the rhGH concentration. In selection of the peptide sequence that shows affinity towards hGH, phage display method was conducted. Additionally the sequences from literature and computational design were used as alternatives. The interaction between these peptides and hGH was investigated by isothermal titration calorimetry and surface plasmon resonance.

In this study, regulatory gene effects on recombinant human growth hormone (rhGH) production by Bacillus subtilis were investigated. For this purpose, firstly Bacillus strains, which are deficient in abrB, aprE, degQ, degS, degU, scoC, sinI, sinR, and spo0A genes, were selected according to the regulatory gene network of aprE gene (serine alkaline protease gene of B. subtilis) since due to the degQ promoter and the pre-signal sequence of subC gene cloned in front of the hGH gene, hGH is produced by mimicking the serine alkaline protease synthesis. R-Bacillus strains were constructed by transformation of pMK4::pre(subC)::hGH plasmid to the selected strains. Thereafter, by the laboratory scale experiments, strains having the highest hGH production capacity were determined as scoC, aprE, sinR, and degU knockout strains. Using these strains, fermentation experiments were carried out in pilot-scale bioreactor in defined medium. Effect of pH control was also investigated and the highest cell and hGH concentration was obtained by scoC knockout strain in pH controlled operation as 1.62 kg m-3 and 126 g m-3, respectively. By this strain, the overall product and cell yield on total substrate were found as 16.12 g kg-1 and 0.15 g g-1, respectively. Furthermore, the highest total protease activity was attained by degU knockout strain as 65 U cm-3. On the other hand, maximum total organic acid secretion was determined as 1.31 kg m-3 in aprE knockout strain.

In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreactor operations were designed and per-formed. In general, the designed strategies are fundamentally based on simulta-neous feeding of the two substrates starting at t=0 h of the production phase, i.e., batch-wise 50 gL-1sorbitol feeding, together with fed-batch methanol feeding with a specific growth rate of &mu / 0=0.03 h-1 or &mu / 0=0.04 h-1, and fed-batch sorbitol feeding with a specific growth rate of &mu / 0=0.025h-1 which was calculated based on the specific consumption rate qS=0.152 g g-1h-1 of sorbitol. Consequently, sorbitol concentration was kept constant at 50 gL-1 within t=0-15h of the production phase / where, sorbitol feeding was terminated at t=15h. Amongst, in the first strategy (SSM1), methanol was fed to the system with the specific growth rate of &mu / 0=0.03 h-1, and the H+ concentration (pH) in the bioreactor was kept constant at pH=5.0. In the second strategy (SSM2), pH was kept constant at 5.5 until t=24h of the induction phase (production phase), thereafter, was reduced to pH= 5.0 / where methanol was fed to the bioreactor with the specific growth rate of &mu / 0=0.03 h-1. In the third strategy (SSM3), methanol was fed with the specific growth rate of &mu / 0=0.04 h-1, and the pH in the bioreactor was kept constant at pH 5.0. The highest rhGH production and cell concentration were achieved in the first strategy SSM1 as CrhGH=640 mg L-1 and CX=105.3 g L-1, and the overall cell and product yields on total substrate were calculated as YX/S =0.21 g g-1 and YCrhGH/S =1.83 mg g-1. In the second part of this study the two-substrates sorbitol and methanol were fed simultaneously in a solution compose of 1.37 mol sorbitol and 6.21 mol methanol in 13.88 mol water, which is named as SM. In this strategy (SM), the two-substrate solution was fed to the medium with the specific growth rate of &mu / 0=0.03 h-1 on sorbitol until t=30h / thereafter, only methanol was fed to the bio-reactor with the specific growth rate of &mu / 0=0.03 h-1. The highest cell and rhGH concentrations obtained in SM were, respectively, Cx=104.7 g L-1 and CrhGH=124 mg L-1 / and the overall cell and product yields on the total substrate were calcu-lated as YX/S=0.21 g g-1 and YCrhGH/S=0.39 mg g-1. Although the highest cell con-centration obtained at SM is close to that of the SSM1, the rhGH concentration obtained at SM is 5.2-fold lower than that of the strategy SSM1.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Feb. 4, 2010). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Feb. 18, 2010). Includes bibliographical references.

Šio darbo tikslas – įvertinti žmogaus rekombinantinio augimo hormono Biosoma (UAB „Biotechna“, Vilnius, Lietuva) įtaką eksperimentinių gyvūnų (žiurkių) odos paviršinės mechaninės, visos odos mechaninės ir visos odos nudegimo žaizdos gijimui. Tyrimo uždaviniai: mechaniškai sukėlus eksperimentiniams gyvūnams odos paviršiaus iki tinklinio sluoksnio defektą, įvertinti Biosoma poveikį žaizdos gijimui, atliekant žaizdos dugno kolageno tinklo histomorfometrinius matavimus; eksperimento su gyvūnais metu mechaniškai sukėlus visos odos storio defektą, nustatyti Biosoma poveikį žaizdos gijimo greičiui, pagal jos kontūro kitimą; nustatyti temperatūros ir ekspozicijos parametrus siekiant sukelti standartizuotą visos odos nudegimo žaizdos sąlyčio modelį; įvertinti Biosoma įtaką eksperimentinių gyvūnų visos odos nudegimo žaizdos gijimui bei palyginti jį su visos odos mechanines žaizdos gijimu; įvertinti Biosoma įtaką gyvūnų visos odos nudegimo gijimui atliekant žaizdos dugno kolageno tinklo histomorfometrinius matavimus. Mūsų tyrimo duomenimis, eksperimentinių gyvūn���� (žiurkių), gavusių sisteminį anabolinį preparatą – žmogaus rekombinantinį augimo hormoną Biosoma 5,6 TV (2,0 mg/kg per parą injekcijas) visų tipų žaizdos – odos paviršinė mechaninė, visos odos mechaninė ir visos odos nudegimo – gijo greičiau, jos dugno vienas pagrindinių struktūrinių komponentų – fibrilinio kolageno tinklas buvo sintetinamas ir „brendo“ greičiau. / The aim of this study is to estimate the influence of recombinant human growth hormone Biosoma (UAB „Biotechna“, Vilnius, Lietuva) on different types of rats skin wound healing. We examined part and full thickness skin mechanical lesions and full thickness skin burns. The objectives: to estimate recombinant human growth hormone Biosoma impact on mechanically inflicted animal part thickness cutaneous wound healing according to histomorphometrical evaluation of the wound bed collagen; to evaluate recombinant human growth hormone Biosoma influence on full thickness skin mechanical wound healing in accordance with the wound contour changing; to choose the thermal exposition parameters for inflicting standard full thickness contact skin burn in animal; to evaluate the influence of recombinant human growth hormone Biosoma on animal full thickness skin burn healing and to compare it with the full thickness skin mechanical wound healing; to estimate the influence of recombinant human growth hormone Biosoma on animal full thickness burn ECM synthesis according to histomorphometrical wound bed collagen measurements. According to the research data, all types skin wounds (part and full thickness skin mechanical and full thickness burn) healed faster, their wound bed collagen fibers synthesis and maturation were faster in the experimental animals (rats) treated with rhGH Biosoma 5.6 IU (2.0 mg/kg a day) injections.