Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant
详细信息 查看全文
- 作者：Sarah Zernia ; Florian Ott ; Kathrin Bellmann-Sickert ; Ronny Frank ; Marcus Klenner ; Heinz-Georg Jahnke ; Andrea Prager ; Bernd Abel ; Andrea Robitzki ; Annette G. Beck-Sickinger
- 刊名：Bioconjugate Chemistry
- 出版年：2016
- 出版时间：April 20, 2016
- 年：2016
- 卷：27
- 期：4
- 页码：1090-1097
- 全文大小：363K
- 年卷期：0
- ISSN：1520-4812
文摘
Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.