Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3.
Recombinant Arabidopsis thaliana (At) RGL-3, using two vectors pMAL-c2 and pET 21, was expressed as inclusion bodies in Escherichia coli under a range of temperature conditions. Only low levels (8-12% of total protein) of soluble protein were produced. The "soluble" fraction was shown by native PAGE to exist as soluble aggregates of RGL-3. A method was developed, consisting of induction of expression at various temperatures that yielded high levels of refoldable inclusion bodies using the pET vector. (At) RGL-3, as inclusion bodies, was solubilized in 8M urea and refolding was initiated by 20-fold direct dilution of denaturant. Under optimal conditions, 87% of the denatured protein of inclusion bodies was successfully re-natured. Refolding was monitored by "native" PAGE. Refolded RGL-3 was shown to be present as monomers and dimers. Attempts to further purify His-tagged RGL-3 using Ni/NTA chromatography resulted in the formation of higher polymers.
Al-Samarrai
Taha H
TH
The Horticulture and Food Research Institute of New Zealand Ltd., Private Bag 11030, Palmerston North, New Zealand. Tal-Samarrai@hortresearch.co.nz
Kirk
Christopher A
CA
Jones
William T
WT
Harvey
Dawn
D
Sun
Xiaolin
X
D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D029681:Arabidopsis Proteins; C485889:RGL3 protein, Arabidopsis; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors