Dihydrofolate reductase as a new "affinity handle".
Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.
Iwakura
M
M
Research Institute for Polymers and Textiles, Ibaraki.
Furusawa
K
K
Kokubu
T
T
Ohashi
S
S
Tanaka
Y
Y
Shimura
Y
Y
Tsuda
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D008727:Methotrexate / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate