Synthesis of 32P-labelled protein probes using a modified thioredoxin fusion protein expression system in Escherichia coli.
The thioredoxin fusion protein expression system from invitrogen was modified so that 32P-labelled recombinant proteins can be easily obtained in large quantities for functional studies. Proteins that are prone to form the inclusion bodies can be functionally expressed as thioredoxin fusion proteins in Escherichia coli. After expression, the recombinant proteins can be easily phosphorylated with 32P-gamma ATP and the 32P-labelled protein can be obtained functionally via a mild proteolytic digestion to cleave off the thioredoxin moiety. A deletion construct of the Ah receptor nuclear translocator protein was used as an example to illustrate how this protein expression system works.
Delucchi
Anthony B
AB
Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, 751 Brookside Road, Stockton, CA 95211, USA.
Jensen
Kyle A
KA
Chan
William K
WK
D000595:Amino Acid Sequence; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D015335:Molecular Probes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013879:Thioredoxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D015335:Molecular Probes; D010761:Phosphorus Radioisotopes; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D013879:Thioredoxins