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- Title
A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins.
- Authors
Masuda, Junko; Takayama, Eiji; Satoh, Ayano; Kojima-Aikawa, Kyoko; Suzuki, Kimihiro; Matsumoto, Isamu
- Abstract
Compared to glutathioneS-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 intoEscherichia coliDH5a, 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 anyEscherichia colistrain; and 3) it can be used in a single strain ofEscherichia coliin all steps from plasmid construction to the expression of the target gene.
- Subjects
GENETIC vectors; GENE expression; MOLECULAR cloning; PROTEINS; GENETIC engineering; BIOTECHNOLOGY
- Publication
Biotechnology Letters, 2004, Vol 26, Issue 20, p1543
- ISSN
0141-5492
- Publication type
Article
- DOI
10.1023/B:BILE.0000045650.90384.b2