One-step chromatographic purification procedure of a His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein overexpressed in Escherichia coli as a secreted form.
A His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein was overexpressed in E. coli as a secreted form in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several hundred milliliters of culture, a centrifugation was used to eliminate the cells. After solubilization and centrifugation, the protein was then purified by a one-step chromatographic purification procedure. Immobilized Metal Affinity Chromatography (IMAC) was performed by evaluating the tri-dentate iminodiacetic acid (IDA) chelating group with chelating Sepharose fast flow, and the tetra-dendate nitrilotriacetic acid (NTA) chelating group with NTA-agarose. The latter was the most suitable gel for our protein. This expression system and the use of affinity chromatography is a rapid technique to obtain a soluble protein for use in structural studies to further understand the mechanisms of HTLV-1 entry into target cells.
Tallet
B
B
Laboratoire de Virologie, Université Victor Segalen Bordeaux 2, France. beatrice.tallet@reger.u-bordeaux2.fr
Astier-Gin
T
T
Castroviejo
M
M
Santarelli
X
X
D002614:Chelating Agents / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015686:Gene Products, env / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015682:Retroviridae Proteins, Oncogenic / Q000737:chemistry*
D002614:Chelating Agents; D015686:Gene Products, env; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; C055266:gp46 protein, Human T-cell leukemia virus type I; D006639:Histidine