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Volumn 277, Issue 5334, 1997, Pages 1994-1996

Identification of a naturally occurring peroxidase-lipoxygenase fusion protein

Author keywords

[No Author keywords available]

Indexed keywords

HYBRID PROTEIN; LIPOXYGENASE; PEROXIDASE;

EID: 0030796446     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5334.1994     Document Type: Article
Times cited : (101)

References (34)
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    • note
    • For the first round of PCR, we used cDNA from P. homomalla total RNA and the primers 5′-GGTTCCAARTGGYTNATGGCNAA and 5′-CTATGTRTGIATRCTRTTIGGIAT (7). For the second round of PCR, we used the equivalent of 0.1 μl of the first-round PCR products as template with the same upstream primer and the nested downstream primer 5′-CCAGATCAGIAAICCRTCRTCICKRTA. The 405-bp product included sequences from the previously identified 8R-lipoxygenase (7) and the lipoxygenase described here.
  • 15
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    • The 5′ end of the sequence was cloned with the Marathon cDNA Amplification kit (Clontech) (7) and the 5′ RACE procedure (Gibco-BRL)
    • The 5′ end of the sequence was cloned with the Marathon cDNA Amplification kit (Clontech) (7) and the 5′ RACE procedure (Gibco-BRL).
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    • 4 tag) was expressed in the pET3a vector in Escherichia coli (BL21, Novagen) incubated at 28°C in TB medium for 24 hours. This resulted in appearance in the bacterial cytosolic fraction of a hemoprotein detectable by UV-visible spectroscopy (≈1 absorbance unit at 406 nm) that was not present in cells transfected with vector alone. The enzyme was purified by ammonium sulfate precipitation (30 to 55% fraction) and by anion exchange chromatography on Q-Sepharose, and on a nickel affinity column (Qiagen) for the His-tagged protein.
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    • We thank W. Boeglin for help with the HPLC analyses and J. Swanson and his colleagues at the Keys Marine Laboratory for collection of the P. homomalla specimens. Supported by a grant from NIH (GM49502) and a Fogerty International Research Collaboration Award (TW00404)
    • We thank W. Boeglin for help with the HPLC analyses and J. Swanson and his colleagues at the Keys Marine Laboratory for collection of the P. homomalla specimens. Supported by a grant from NIH (GM49502) and a Fogerty International Research Collaboration Award (TW00404).


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