High-level expression of recombinant pea chloroplast fructose-1,6-bisphosphatase and mutagenesis of its regulatory site.
The cDNA fragment coding for mature chloroplast pea fructose-1,6-bisphosphatase [Fru(1,6)P2ase] was introduced by PCR into the expression vector pET-3d resulting in the construction pET-FBP. After transformation of BL21 (DE3) Escherichia coli cells by the pET-FBP plasmid and induction with isopropyl thio-beta-D-galactoside, high-level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P2ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three-dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.
Jacquot
J P
JP
Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France.
Lopez-Jaramillo
J
J
Chueca
A
A
Cherfils
J
J
Lemaire
S
S
Chedozeau
B
B
Miginiac-Maslow
M
M
Decottignies
P
P
Wolosiuk
R
R
Lopez-Gorge
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005809:Genes, Regulator; D008027:Light; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry; D018532:Pisum sativum / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins
D009841:Oligonucleotides; D011994:Recombinant Proteins; D006597:Fructose-Bisphosphatase