A new protocol for high-yield purification of recombinant human prothymosin alpha expressed in Escherichia coli for NMR studies.
Human prothymosin alpha (ProTalpha) is a small acidic protein (12.1 kDa; pI approximately 3.5) ubiquitously expressed in a wide variety of tissues. The amino acid composition of this protein is highly unusual. While close to half of its sequence is composed of acidic amino acids, the protein does not contain any aromatic residues. ProTalpha has been shown to play crucial roles in different biological processes including cell proliferation, transcriptional regulation and apoptosis. Despite the multiple functions this protein has, it does not adopt a stable tertiary fold under physiological conditions. In order to understand how ProTalpha functions, detailed structural characterization of this protein is essential. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for elucidating the protein structure and dynamics at the atomic level. However, milligrams of isotopically labeled protein with high purity are usually required for the studies. In this work, we developed a high-yield protocol for purifying recombinant ProTalpha expressed in Escherichia coli by exploiting the intrinsically disordered and acidic natures of this protein. By combining the heat-cooling extraction, ammonium sulfate precipitation, and anion exchange chromatography, we were able to obtain over 20 mg of ProTalpha with >97% purity from 1L of M9 minimal media culture. The new purification protocol provides a cost effective and an efficient way to produce large quantities of high purity recombinant human ProTalpha in various isotopically labeled forms, which will greatly facilitate the structural studies of this protein by NMR and other biophysical methods.
Yi
Shiluan
S
Department of Biochemistry, The University of Western Ontario, London, Ont., Canada N6A 5C1.
Brickenden
Anne
A
Choy
Wing-Yiu
WY
D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic; D000645:Ammonium Sulfate / Q000737:chemistry; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014461:Ultracentrifugation
D024342:Amino Acids, Acidic; D011498:Protein Precursors; D011994:Recombinant Proteins; C040782:prothymosin alpha; D013947:Thymosin; D000645:Ammonium Sulfate