Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose.
A natural 19-amino-acid poly-histidine affinity tag was cloned at the N-terminus of three recombinant proteins. The vectors containing the DNA of the fusion proteins were used for transformation of Escherichia coli DH5alpha cells. Each protein was expressed, extracted and purified in one chromatographic step. The purification procedure for each protein can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent--Co2+-carboxymethylaspartate agarose Superflow--was utilized at linear flow-rates as high as 5 cm/min. The final preparation of each protein is with purity greater than 95% as ascertained by sodium dodecyl sulfate-electrophoresis. Recovery for each purified protein was higher than 77% of the initial loaded amount as judged by biological activity. The operational capacity of Co2+-carboxymethylaspartate agarose for each protein was determined.
Chaga
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Clontech Laboratories, Inc., Palo Alto, CA 94303, USA.
Bochkariov
D E
DE
Jokhadze
G G
GG
Hopp
J
J
Nelson
P
P
D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006639:Histidine; D008164:Luminescent Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D008164:Luminescent Proteins; D010455:Peptides; D011994:Recombinant Proteins; C070802:carboxymethylaspartic acid agarose; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D001224:Aspartic Acid; D006639:Histidine; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase