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Effect of Polymer Shielding on Elution of G3PDH Bound to Dye-ligand Adsorbent
Ling Tau Chuan,Lyddiatt Andrew The Korean Society for Biotechnology and Bioengine 2006 Biotechnology and Bioprocess Engineering Vol.11 No.1
Batch binding experiments were performed to assess the recovery performance of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) bound to the unshielded and polymer (polyvinyl pyrrolidone. PVP)-shielded dye-ligand (Cibacron Blue 3GA) adsorbent. The adoption of a polymer-shielded, dye-ligand technique facilitated the elution efficiency of bound G3PDH. It was demonstrated that the recovery of G3PDH using polymer-shielded dye-ligand adsorption yielded higher elution efficiency, at 60.5% and a specific activity of 42.3 IU/mg, after a low ionic strength elution (0.15 M NaCl). The unshielded dye-ligand yielded lower elution efficiency. at 6.5% and a specific activity of 10.2 IU/mg.
Ling Tau Chuan,Chow Yen Mei,Tey Beng Ti,Mohammad Nordin Ibrahim,Arbakariya Ariff 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.3
Bakers yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.
Tau Chuan Ling,Yen Mei Chow,Beng Ti Tey,Mohd Nordin Ibrahim,Arbakariya Ariff 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.6
The influence of whole yeast cells (0~15% w/v) on the protein adsorption performance in dye-ligand chromatography was explored. The adsorption of a model protein, bovine serum albumin (BSA), was selected to demonstrate this approach. The UpFront adsorbent (ρ = 1.5 g/cm3) derivatised with Cibacron Blue 3GA and a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography, Denmark, were employed in the batch binding and expanded bed operation. The BSA binding capacity was demonstrated to not be adversely affected by the presence of yeast cells. The dy-namic binding capacity of BSA at a C/C0 = 0.1 biomass concentration of 5, 10, 15% w/v were 9, 8, and 7.5 mg/mL of settled adsorbent, respectively.
Purification of Filamentous Bacteriophage M13 by Expanded Bed Anion Exchange Chromatography
Tau Chuan Ling,Chee Kin Loong,Wen Siang Tan,Beng Ti Tey,Wan Mohammad Wan Abdullah,Arbakariya Ariff 한국미생물학회 2004 The journal of microbiology Vol.42 No.3
In this paper, we investigated the development of a simplified and rapid primary capture step for the recovery of M13 bacteriophage from particulate-containing feedstock. M13 bacteriophage, carrying an insert, was propagated and subsequently purified by the application of both conventional multiple steps and expanded bed anion exchange chromatography. In the conventional method, precipitation was conducted with PEG/NaCl, and centrifugation was also performed. In the single step expanded bed anion exchange adsorption, UpFront FastLineTM20 (20 mm i.d.) from UpFront Chromatography was used as the contactor, while 54 ml (Ho=15cm) of STREAMLINE DEAE (ρ=1.2 g/cm3) from Amersham Pharmacia Biotechnology was used as the anion exchanger. The performance of the two methods were evaluated, analysed, and compared. It was demonstrated that the purification of the M13 bacteriophage, using expanded bed anion exchange adsorption, yielded the higher recovery percentage, at 82.86%. The conventional multiple step method yielded the lower recovery percentage, 36.07%. The generic application of this integrated technique has also been assessed.
Tau Chuan Ling,Yen Mei Chow,Beng Ti Tey,Mohd Nordin Ibrahim,Arbakariya Ariff 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.3
A dense, pellicular UpFront adsorbent (ρ = 1.5 g/cm3, UpFront Chromatography, Cophenhagen, Denmark) was characterized in terms of hydrodynamic properties and protein adsorption performance in expanded bed chromatography. Cibacron Blue 3GA was immobilised into the adsorbent and protein adsorption of bovine serum albumin (BSA) was selected to test the setup. The Bodenstein number and axial dispersion coefficient estimated for this dense pellicular adsorbent was 54 and 1.63 × 10-5 m2/s, respectively, indicating a stable expanded bed. It could be shown that the BSA protein was captured by the adsorbent in the presence of 30% (w/v) of whole-yeast cells with an estimated dynamic binding capacity (C/Co = 0.01) of approximately 6.5 mg/mL adsorbent.
Ling Tau Chuan,Chow Yen Mei,Tey Beng Ti,Mohammad Nordin Ibrahim,Arbakariya Ariff 한국생물공학회 2005 Biotechnology and Bioprocess Engineering Vol.10 No.3
The adsorption of a model protein bovine serum albumin (BSA) in expanded bed chromatography was undertaken by exploiting a commercially available expanded bed column (20 mm i.d.) from UpFront Chromatography and Streamline DEAE ( = 1.2 g/cm3) from Amersham Pharmacia Biotechnology. The influence of whole yeast cells on the adsorption capacity of column was explored by employing yeast cells in a concentration ranged of 0 to 15% (w/v). Equilibrium isotherms for adsorption of BSA on Streamline DEAE were correlated by using Langmuir equation. The presence of yeast cells resulted in decreased of BSA binding capacity in both batch binding and expanded bed chromatography. Results indicated that the yeast cells act as competitor for proteins to bind to the sites on adsorbents.
Effect of Polymer Shielding on Elution of G3PDH Bound to Dye-ligand Adsorbent
Tau Chuan Ling,Andrew Lyddiatt 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.1
Batch binding experiments were performed to assess the recovery performance of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) bound to the unshielded and polymer (polyvinyl pyrrolidone, PVP)-shielded dye-ligand (Cibacron Blue 3GA) adsorbent. The adoption of a polymer-shielded, dye-ligand technique facilitated the elution efficiency of bound G3PDH. It was demonstrated that the recovery of G3PDH using polymer-shielded dye-ligand adsorption yielded higher elution efficiency, at 60.5% and a specific activity of 42.3 IU/mg, after a low ionic strength elution (0.15 M NaCl). The unshielded dye-ligand yielded lower elution efficiency, at 6.5% and a specific activity of 10.2 IU/mg.
The Performance of a Glass Bead Shaking Technique for the Disruption of Escherichia coli Cells
Ramakrishnan Nagasundara Ramanan,Tau Chuan Ling,Arbakariya B. Ariff 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.5
The efficacy of a simple laboratory method for cell disruption based on the shaking of glass beads on a rotary shaker was assessed in this study, via measurements of the release of total protein and interferon-2b from E. coli. The op-timum conditions for cell disruption were detected after 30 min of shaking in Tris-HCl buffer (pH 8) at 300 rpm with 1.5 g of glass beads (diameter: 0.5 mm) per mL of cell suspension volume. Three test runs were conducted under the above conditions and the maximum average protein release values were determined as 3.048, 3.564, and 3.015 mg/mL, respectively. The amount of protein release was comparable to the amount of protein release in ultrasonica-tion and glass bead vortexing procedures. The amount of interferon-2b release in the ultrasonication, glass bead vortexing, and glass bead shaking trials were 240, 172, and 201 ng/mL, respectively. This method was shown to process between 1 and 10 mL of sample volume in a 50 mL Falcon tube without a great deal of deviation, and was able to handle in excess of 60 samples simultaneously.
A Fermentation Strategy for Anti-MUC1 C595 Diabody Expression in Recombinant Escherichia coli
John Chi-Wei Lan,Tau Chuan Ling,Grant Hamilton,Andrew Lyddiatt 한국생물공학회 2006 Biotechnology and Bioprocess Engineering Vol.11 No.5
The development of fermentation conditions for the production of C595 diabody fragment (dbFv) in E. coli HB2151 clone has been explored. Investigations were carried out to study the effect of carbon supplements over the expression period, the comparison of C595 dbfv production in synthetic and complex media, the influence of acetic acid upon antibody production, and comparison of one-stage and two-stage processes operated at batch or fed-batch modes in bioreactor. Yeast extract supplied during expression yielded more antibody fragment than any other carbon supply. The synthetic medium presented higher specific productivity (0.066 mg dbFv g-1 dry cell weight) when compared to the complex medium (0.044 mg dbFv g-1 DCW). The comparison of fermentation strategies demonstrated that (1) one-stage fed-batch fermentation performed higher C595 dbFv production than that operated in batch mode which was significantly affected by acetate concentration; (2) a two-stage batch operation could enhance C595 dbFv production. It was found that a concentration of 12.3 mg L-1 broth of C595 dbFv and a cell concentration of 10.8 g L-1 broth were achieved at the end of two-stage operation in 5-L fermentation.