Premium
Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli
Author(s) -
Rogers W.John,
Hodges Michael,
Decottignies Paulette,
Schmitter Jean-Marie,
Gadal Pierre,
Jacquot Jean-Pierre
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)81340-r
Subject(s) - chlamydomonas reinhardtii , complementary dna , microbiology and biotechnology , ferredoxin , biology , escherichia coli , peptide sequence , recombinant dna , expression vector , coding region , sequence analysis , rapid amplification of cdna ends , oligonucleotide , biochemistry , molecular cloning , gene , enzyme , mutant
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 Nco I and Bam HI 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.