A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins.
Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge. We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no. AB116367), for producing recombinant His-fusion proteins. This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector. Human annexin IV (Anx IV) was used as target protein. His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted. To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3). Using pHisJM, Anx IV protein was highly expressed in all cell strains. In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.
Masuda
Junko
J
Graduate School of Humanities, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
Takayama
Eiji
E
Satoh
Ayano
A
Kojima-Aikawa
Kyoko
K
Suzuki
Kimihiro
K
Matsumoto
Isamu
I
D000595:Amino Acid Sequence; D017314:Annexin A4 / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D017314:Annexin A4; D011993:Recombinant Fusion Proteins; D006639:Histidine