| RRC ID |
55714
|
| 著者 |
Xxxx P, Minamihata K, Tatsuke T, Lee JM, Kusakabe T, Kamiya N.
|
| タイトル |
Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.
|
| ジャーナル |
Biotechnol J
|
| Abstract |
Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.
|
| 巻・号 |
13(6)
|
| ページ |
e1700624
|
| 公開日 |
2018-6-1
|
| DOI |
10.1002/biot.201700624
|
| PMID |
29717548
|
| MeSH |
Animals
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
Biotechnology
Biotin / chemistry
Biotin / metabolism
Biotinylation
Bombyx / genetics
Bombyx / metabolism*
Horseradish Peroxidase / chemistry
Horseradish Peroxidase / genetics
Horseradish Peroxidase / metabolism*
Larva / genetics
Larva / metabolism
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism*
Spectrophotometry, Ultraviolet
Staphylococcal Protein A / chemistry
Staphylococcal Protein A / genetics
Staphylococcal Protein A / metabolism*
Streptavidin / chemistry
Streptavidin / metabolism
|
| IF |
3.543
|
| 引用数 |
2
|
| リソース情報 |
| カイコ |
f38 |
