Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli.
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 (DH5 alpha 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.
Rogers
W J
WJ
Laboratoire de Physiologie Végétale Moléculaire, Université de Paris Sud, UA 1128 CNRS, Orsay, France.
Hodges
M
M
Decottignies
P
P
Schmitter
J M
JM
Gadal
P
P
Jacquot
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry
D004277:DNA, Single-Stranded; D005288:Ferredoxins; D011994:Recombinant Proteins