Expression in Escherichia coli and in vitro refolding of the plant transcription factor Arabidopsis thaliana RGL3

• Published in: Protein expression and purification 2007-06, Vol.53 (2), p.289-292
• Main Authors: Al-Samarrai, Taha H., Kirk, Christopher A., Jones, William T., Harvey, Dawn, Sun, Xiaolin
• Format: Article
• Language: English
• Subjects: Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - isolation & purification; Arabidopsis thaliana; Arabidopsis thaliana RGL3; DELLA family; Dimerization; Escherichia coli; Escherichia coli - genetics; Genetic Vectors; In vitro refolding; Plant proteins; Plant transcriptional factors; Protein Denaturation; Protein Folding; Protein Structure, Quaternary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - isolation & purification
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2007.01.008

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.