title: Phase system selection with fractional factorial design for purification of recombinant cyanovirin-N from a hydroponic culture medium using centrifugal partition chromatography.
abstract: Centrifugal partition chromatography (CPC) with an aqueous two-phase system (ATPS) was used to purify recombinant cyanovirin-N (CV-N) from other proteins which were co-secreted into a hydroponic plant medium in a rhizosecretion process. To achieve satisfactory protein concentration, the purification was preceded by ultrafiltration performed on a 5 kDa filter. ATPS, because of their gentle nature, were selected as the phase system for CPC. A systematic phase system selection was applied. This involved studying the effect of seven parameters of ATPS: polymer type, salt type, the polymer and salt concentration, the polymer molecular weight, pH, and presence of two additional salts; NaCl and NaClO4, which all together gave 320 combinations. design of experiment (DoE) software allowed the reduction of this number to 46. Having tested partitioning of cyanovirin-N and impurities in 46 ATPS, the three best potential phase systems generated by the programme were then tested on the CPC. Out of these three, 13/13% PEG4000 sodium phosphate, pH 3.0, proved to be most effective phase system in the purification of cyanovirin-N, judged by ELISA and SDS-PAGE analysis, as it eliminated most of the impurities from the final cyanovirin-N preparation.
authors.affiliation: Brunel Institute for Bioengineering, Brunel University, London, UK. lukasz.grudzien@brunel.ac.uk
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Grudzień
authors.forename: Luisa
authors.initials: L
authors.lastname: Madeira
authors.forename: Derek
authors.initials: D
authors.lastname: Fisher
authors.forename: Julian
authors.initials: J
authors.lastname: Ma
authors.forename: Ian
authors.initials: I
authors.lastname: Garrard
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002498:Centrifugation / Q000379:methods*; D003377:Countercurrent Distribution / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D018527:Hydroponics; D008970:Molecular Weight; D010472:Perchlorates / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry; D017670:Sodium Compounds / Q000737:chemistry; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D010472:Perchlorates; D011108:Polymers; D011994:Recombinant Proteins; D017670:Sodium Compounds; C107733:cyanovirin N; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C031068:sodium perchlorate