title: Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract.
abstract: Tobacco has been studied as a possible host for the production of recombinant proteins. In this report, recombinant beta-glucuronidase (rGUS) was used as a model protein to study the feasibility of using polyethyleneimine (PEI) precipitation to fractionate acidic recombinant proteins from transgenic tobacco. Results showed that rGUS was preferentially precipitated when the PEI dosage was beyond 200mg PEI/g total protein. At 700-800 mg PEI/g total protein, nearly 100% rGUS was precipitated with less than 40% native tobacco proteins co-precipitated. Approximately 85-90% of the rGUS activity could be recovered from the precipitation pellet for an enrichment ratio of 2.7-3.4. As a comparison, anion exchange chromatography (AEX) yielded similar results to PEI precipitation with 66-90% rGUS activity recovered and an enrichment ratio of 1.8-3.1. The rGUS was further purified by an additional hydrophobic interaction chromatographic (HIC) step after precipitation or AEX. Similar results in terms of rGUS activity recovered and enrichment were obtained. The major interfering protein at the end of all purification steps is most likely the Fraction 1 protein ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). The presence of this protein is likely the cause for the varying and somewhat low enrichment ratios, but it may be later removed via a size-exclusion chromatography step. PEI precipitation offers the advantage of allowing significant sample concentration prior to subsequent purification techniques such as chromatography and is much less expensive than chromatographic methods as well. Through direct comparison, this study shows that PEI may be used as an initial fractionation step in replacement of AEX to facilitate the purification of acidic recombinant proteins from transgenic tobacco.
authors.affiliation: Department of Biological Systems Engineering, 210 Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Holler
authors.forename: David
authors.initials: D
authors.lastname: Vaughan
authors.forename: Chenming
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005966:Glucuronidase / Q000302:isolation & purification*; D030821:Plants, Genetically Modified / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D005966:Glucuronidase