Laboratory-Scale Hydroxylation of Steroids by P450BM3 Variants
| Autor: | Nathalie Nett, Sabrina Hoebenreich, Martin Spinck |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Plasmid preparation Lysis medicine.disease_cause Enzyme catalysis law.invention Hydroxylation 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Column chromatography Biochemistry Biotransformation chemistry law medicine Recombinant DNA Escherichia coli |
| Zdroj: | Microbial Steroids ISBN: 9781493971824 |
| DOI: | 10.1007/978-1-4939-7183-1_17 |
| Popis: | This chapter describes the asymmetric hydroxylation of steroids on laboratory preparative scale, using engineered variants of P450BM3 (CYP102A1) as enzyme catalyst. The following protocol covers the creation of an Escherichia coli BL21-Gold (DE3) expression strain, including necessary control experiments like plasmid preparation, test expression, and creation of storage cultures, to verify successful experimental access to recombinant expressed P450BM3 variants. The recombinant expressed P450BM3 variants are obtained as cleared cell lysate and used in a biotransformation setup to hydroxylate 2.8 mg and up to 15 mg testosterone in the presented protocol. Since P450BM3 depends on NADPH as an electron source for the reaction, a glucose and glucose dehydrogenate based recycling system is added to the reaction. The protocol further includes liquid-liquid extraction of hydroxytestosterone and directs the experimenter to compound purification via column chromatography. |
| Databáze: | OpenAIRE |
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