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Functional Expression and Purification of CYP93C20，a Plant Membrane-Associated Cytochrome P450 from Medicago truncatula.

Abstract：

:Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coli XL1-blue cells and was purified by Ni(2+)-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Chang Z,Wang X,Wei R,Liu Z,Shan H,Fan G,Hu H

doi

10.1016/j.pep.2010.11.012

subject

Has Abstract

pub_date

2010-12-04 00:00:00

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(10)00322-0

pub_type

杂志文章

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