Refolding and one-step purification of recombinant human ARA70 over-expressed in Escherichia coli.
Androgen receptor (AR)-associated coregulator 70 (ARA70) is a cytoplasmic protein that has been characterized to have the ability to induce AR transcriptional activity in response to androgens and anti-androgens in prostate cancer cells. AR has been shown to have an important role in the progression of prostate cancer and in normal male reproductive system development. To elucidate the molecular mechanisms and biological relevance of ARA70 to prostrate cancer using a variety of biochemical analyses, the cDNA encoding full length ARA70 was cloned into pET21b vector. Here, we report the refolding and one-step purification of ARA70 from inclusion bodies over-expressed in Escherichia coli. The protein was purified to homogeneity, yielding approximately 60 mg ARA70 from 1L of terrific broth media. Refolding process of ARA70 was monitored using far-UV CD analysis.
Singh
Vinay Kumar
VK
Department of Biochemistry, Queen's University, Kingston, Ont., Canada K7L 3N6.
Jia
Zongchao
Z
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D008969:Molecular Sequence Data; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014170:Transformation, Genetic
C111382:NCOA4 protein, human; D056918:Nuclear Receptor Coactivators; D015513:Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors