title: Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems.
abstract: The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-1)) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.
authors.affiliation: Biochemical Recovery Group, Centre for Formulation Engineering, Department of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. ltc555@eng.upm.edu.my
authors.forename: Tau Chuan
authors.initials: TC
authors.lastname: Ling
authors.forename: Andrew
authors.initials: A
authors.lastname: Lyddiatt
mesh_terms: D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D004396:Coloring Agents; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000302:isolation & purification* / Q000378:metabolism*; D008024:Ligands; D044085:Microfluidics / Q000379:methods; D010812:Physical Stimulation / Q000379:methods; D011108:Polymers; D012441:Saccharomyces cerevisiae / Q000302:isolation & purification* / Q000378:metabolism*; D018511:Systems Integration
chemical_list: D004396:Coloring Agents; D008024:Ligands; D011108:Polymers; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)