Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli

FEBS Letters

Decottignies, Paulette²  Schmitter, Jean-Marie¹  Hodges, Michael²  Gadal, Pierre²  Jacquot, Jean-Pierre²  Rogers, W.John² 

[1] Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France;Laboratoire de Physiologie, Végétale Moléculaire, Université de Paris Sud, UA 1128 CNRS, Bâtiment 430, 91405 Orsay-Cedex, France

关键词: Ferredoxin;    PCR;    Chlamydomonas reinhardtii;   

DOI  :  10.1016/0014-5793(92)81340-R

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5α F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.

【 授权许可】

Unknown