Pichia pastoris ile rekombinant insan büyüme hormonu üretimi ve peptit ligandlarla etkileşiminin incelenmesi.

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°C, there was no difference, at 27 and 25°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°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 μ=0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at μ=0.04 h-1, the highest rhGH concentration was achieved at μ=0.03 h-1. Secondly, conducting methanol feeding at μ=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.M.S. - Master of Scienc

Dynamic flux balance analysis for pharmaceutical protein production by Pichia pastoris: Human growth hormone

The influence of methanol feeding rate on intracellular reaction network of recombinant human growth hormone (rhGH) producing Pichia pastoris was investigated at three different specific growth rates, namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04h(-1) (MS-0.04) where Period-I (33 51 h) is the diminution phase for rhGH and cell synthesis. In Period-I, almost all of the formaldehyde entered the assimilatory pathway, at MS-0.02 and MS-0.03, whereas, at MS-0.04 high methanol feeding rate resulted in an adaptation problem. In Period-III, only at MS-0.02 co-carbon source sorbitol uptake-flux was active showing that sorbitol uptake does not affected from the predetermined feeding rate of methanol at mu(0) > 0.02 h(-1). The biomass synthesis flux value was the highest in Period-I, -II and -III, respectively at MS-0.03 & MS-0.04, MS-0.04 and MS-0.02; whereas, rhGH flux was the highest in Period-I, -II, and -III, respectively at MS-0.03, MS-0.02 and MS-0.03. Based on the fluxes, Period-I should start with MS-0.03 methanol feeding rate and starting from the middle of Period-II methanol feeding rate should be shifted to MS-0.02. (c) 2010 Elsevier Inc. All rights reserved

Inhibition of infection spread by co-transmitted defective interfering particles

<div><p>Although virus release from host cells and tissues propels the spread of many infectious diseases, most virus particles are not infectious; many are defective, lacking essential genetic information needed for replication. When defective and viable particles enter the same cell, the defective particles can multiply while interfering with viable particle production. Defective interfering particles (DIPs) occur in nature, but their role in disease pathogenesis and spread is not known. Here, we engineered an RNA virus and its DIPs to express different fluorescent reporters, and we observed how DIPs impact viral gene expression and infection spread. Across thousands of host cells, co-infected with infectious virus and DIPs, gene expression was highly variable, but average levels of viral reporter expression fell at higher DIP doses. In cell populations spatial patterns of infection spread provided the first direct evidence for the co-transmission of DIPs with infectious virus. Patterns of spread were highly sensitive to the behavior of initial or early co-infected cells, with slower overall spread stemming from higher early DIP doses. Under such conditions striking patterns of patchy gene expression reflected localized regions of DIP or virus enrichment. From a broader perspective, these results suggest DIPs contribute to the ecological and evolutionary persistence of viruses in nature.</p></div

Co-propagation of infectious and defective virus.

<p>(a) Experimental setup. Cells were infected with a high MOI (30) of infectious virus and varying amounts of DIP-GFP(0-84). These infected cells were mixed with a large excess of uninfected cells, plated, and the spread progress of infectious and defective virus was observed via time-lapse microscopy. (b) Representative spreading infections. The first four columns contain merged images with red showing infectious virus expression, green showing defective virus expression, and yellow showing areas of both infectious and defective virus expression. The last two columns separate the red and green expression from the 25 hours post infection (hpi) images. The scale bar is 0.5 mm. (c) Infectious virus spread rate (<i>μ</i>m/h) as a function of DIP-GFP input. Individual plaques shown as points, the average as the line. (d) The normalized intensity during the earliest detectable spread, near plaque centers, for infectious (red points and line) and defective (green points and line) virus versus DIP-GFP input.</p

Structure and function of natural and engineered viruses.

<p>(a) Replication potential of natural infectious virus and defective interfering particles (DIPs). An infectious virus alone cannot replicate, but after it infects a permissive cell, viral progeny are produced. A DIP alone can enter a cell, but it cannot replicate. However, when an infectious virus and DIP infect the same cell, DIPs can replicate at the expense of infectious virus. Here figures highlight qualitative relationships between inputs and outputs. (b) Structure of natural and engineered virus genomes. (c) Production of particles, total and infectious (PFU), depends on level of natural (DIP) or engineered (DIP-GFP) input to co-infected cells. (d) Expression of virus or DIP reporter depends on level of natural (DIP) or engineered (DIP-GFP) inputs to co-infected cells. Values are normalized to a no-DIP control (PFU, RFP) or highest DIP input (GFP).</p

Single-cell measures of reporter expression show trade-offs between infectious and defective virus.

<p>(a) Experimental setup. Cells in all conditions were infected with a constant input of infectious virus (30 particles/cell) and varying amounts of DIP-GFP(0-to-84 particles/cell). Individual cells were imaged by time-lapse fluorescence microscopy, as detailed in Methods. For each tracked cell kinetic parameters were estimated for each RFP and GFP expression profile. (b) Example single cell kinetics. RFP and GFP kinetics are shown for two representative cells for DIP input levels 0 and 10. The average RFP and GFP expressions are also shown (dark red and green lines). (c) Anticorrelation between infectious and defective virus yields in single cells. Each gray point is an individual cell, and the green and red lines represent average defective and infectious virus yields respectively.</p

Spatial analysis of defective and infectious virus spread.

<p>(a) Example image (DIP-GFP input = 10) of a plaque maximum intensity projection with concentric rings overlayed, where the center is the plaque origin and each ring has a width of 100 pixels (116 <i>μ</i>m). (b) Example ring (r = 5) from the maximum intensity projection shown in (a). (c) The infectious virus (RFP) expression versus defective virus (GFP) expression for all pixels in (b). The color corresponds to point density with red being the highest and blue the lowest. The gray lines gate positive and negative populations. (d) The infectious virus (RFP) expression versus defective virus (GFP) expression for selected rings of the plaque; here, all pixel intensities that fall below both reporter thresholds have been excluded. For each ring, the contribution of each virus sub-population (e.g., infectious, defective or both) is shown as a percentage of the total population.</p

Effect of co-substrate sorbitol different feeding strategies on human growth hormone production by recombinant Pichia pastoris

BACKGROUNDEffects of co-substrate sorbitol different feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut(+) were investigated by eight designed experiments grouped as: (i) fed-batch methanol feeding without the co-substrate; (ii) fed-batch methanol feeding with pulse sorbitol feeding; (iii) fed-batch methanol feeding together with fed-batch sorbitol feeding at t=0-15h, followed by fed-batch methanol feeding; and (iv) fed-batch methanol and sorbitol feeding at t=0-30h, followed with fed-batch methanol feeding

Fermentation and oxygen transfer characteristics in recombinant human growth hormone production by Pichia pastoris in sorbitol batch and methanol fed-batch operation

BACKGROUND: The influence of methanol feed rate on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut(+) in medium containing sorbitol was investigated at three different specific growth rates (mu), namely, 0.02 (MS-0.02), 0.03 (MS-0.03), and 0.04 (MS-0.04)