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J. Biochem, 2003, Vol. 134, No. 3 403-413
© 2003 Japanese Biochemical Society

MOLECULAR BIOLOGY

High-Level Expression of Porcine Liver Cytochrome P-450 Reductase Catalytic Domain in Escherichia coli by Modulating the Predicted Local Secondary Structure of mRNA

Shigenobu Kimura* and Takashi Iyanagi

Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kouto 3-2-1, Kamigori, Hyogo 678-1297

A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori+. PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr60Ser61Ser62 in PsCPR with those for Ala60Pro61Pro62 in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr60Ser61Ser62 codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr58 and Thr60Ser61 in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by introducing silent mutations at the codons for Thr60Ser61 in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8 mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.

* To whom correspondence should be addressed. Tel: +81-791-58-0207, Fax: +81-791-58-0132, E-mail: s-kimura{at}sci.himeji-tech.ac.jp


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S. Kimura, T. Umemura, and T. Iyanagi
Two-Cistronic Expression Plasmids for High-Level Gene Expression in Escherichia coli Preventing Translational Initiation Inhibition Caused by the Intramolecular Local Secondary Structure of mRNA
J. Biochem., April 1, 2005; 137(4): 523 - 533.
[Abstract] [Full Text] [PDF]


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