EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT N-EPSILON-METHYLTRANSFERASE/
Q. Zheng et al., EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE LARGE SUBUNIT N-EPSILON-METHYLTRANSFERASE/, Protein expression and purification (Print), 14(1), 1998, pp. 104-112
Citations number
18
Categorie Soggetti
Biochemical Research Methods",Biology,"Biothechnology & Applied Migrobiology
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subuni
t (LS) N-6-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes me
thylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Pr
ogress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but
most of the expressed protein was found in the insoluble fraction as
an inclusion body. Expression at lower temperatures increased the leve
l of soluble Rubisco LSMT and the associated enzymatic activity. Howev
er, the soluble form of Rubisco LSMT occurred as two molecular mass fo
rms with the lower molecular mass suggestive of N-terminal processing
at Ser-37, Deletion of 108 nucleotides from the 5' end encoding the N-
terminal 38 amino acids of Rubisco LSMT resulted in a 16-fold increase
in solubility and activity. Further addition of a 3' nucleotide seque
nce coding for a hexahistidyl carboxy-terminal peptide enabled purific
ation of the N-terminally truncated Rubisco LSMT to homogeneity. Five
milligrams of pure recombinant Rubisco LSMT was obtained from a l-lite
r E. coli cell culture. The apparent kinetic constants for recombinant
Rubisco LSMT for spinach Rubisco and AdoMet were only slightly differ
ent from the constants determined using affinity-purified native Rubis
co LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduc
tion in the kappa(cat) for Rubisco LSMT, which was apparently a conseq
uence of catalytic inactivation due to exposure to NiSO4 during purifi
cation. The availability of larger quantities of purified Rubisco LSMT
should enable studies of the structure-function relationships in Rubi
sco LSMT and moreover its interaction with Rubisco. (C) 1998 Academic
Press.