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Cloning and expression of Cryptochrome proteins from Sunflower
Author(s) -
Vercellino Justin,
Dyer James
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.887.16
Subject(s) - cryptochrome , biology , fusion protein , sunflower , helianthus annuus , complementary dna , thaumatin , genetics , circadian clock , microbiology and biotechnology , biochemistry , gene , recombinant dna , agronomy
Two cryptochrome proteins, CRY1 and CRY2, occur in plants and animals. CRY1 regulates the circadian clock in a light‐dependent fashion in insects and plants. Also in plants, blue light photoreception can be used to cue developmental signals. Despite much research on the topic, the specific way that the pterin and flavin chromophores function in these proteins is still poorly understood. The goal of this research was to clone and express the Helianthus annuus (Sunflower) CRY1 protein in an E. coli expression system. Consensus sequence primers flanking the complete coding region based on known sequences of other dicot plant cryptochromes were used in a 5'and 3' Rapid Amplification of cDNA Ends protocol, and the complete CRY1 sequence cloned into a pDEST vector. The sunflower CRY1 was expressed with N‐terminal GST and His tags. The expressed fusion proteins were purified using Glutathione agarose or Ni‐NTA affinity chromatography. Purified proteins will be used for structural and functional analysis.