Genetically engineered polymers of human CuZn superoxide dismutase. Biochemistry and serum half-lives.
CuZn superoxide dismutase is a highly stable dimer of identical subunits with a combined molecular mass of 32,000 daltons. Two human superoxide dismutase genes have been joined in the same translational reading frame, using spacers of different lengths, to encode single chain proteins consisting of two identical human superoxide dismutase subunits. The first construct encodes two directly linked subunits; the terminal glutamine codon of the first gene was changed to a methionine codon and followed immediately by the second gene. The second construct encodes two subunits linked by a 19-amino-acid human immunoglobulin IgA1 hinge sequence. Both constructs produce high levels of catalytically active superoxide dismutase when expressed in Escherichia coli. The protein containing the IgA1 hinge sequence forms polymers up to 750,000 in molecular weight, which are linked together noncovalently by the hydrophobic bonding of the dimer interface. The polymers are soluble, thermostable, and of near normal specific activity. Site-directed in vitro mutagenesis was used to inactivate one of the two human superoxide dismutase subunits. The resulting human superoxide dismutase polymers have approximately 50% activity, thus confirming that the products of both genes are catalytically active. Large amounts of individual polymeric forms have been purified from recombinant yeast and tested for serum stability in rats. The serum half-life is approximately 7 min for both the two-chain wild type human superoxide dismutase dimer (Mr 32,000) and the single chain molecule consisting of a human superoxide dismutase dimer covalently linked by the immunoglobulin hinge region (Mr 34,000), whereas the higher molecular weight polymers (Mr greater than or equal to 68,000) all have half-lives of approximately 145 min.
Hallewell
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RA
Chiron Corporation, Emeryville, California 94608.
Laria
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I
Tabrizi
A
A
Carlin
G
G
Getzoff
E D
ED
Tainer
J A
JA
Cousens
L S
LS
Mullenbach
G T
GT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003461:Crystallography; D004274:DNA, Recombinant / Q000302:isolation & purification; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005813:Genes, Synthetic; D006207:Half-Life; D006801:Humans; D007070:Immunoglobulin A / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics* / Q000302:isolation & purification / Q000493:pharmacokinetics
D004274:DNA, Recombinant; D007070:Immunoglobulin A; D046911:Macromolecular Substances; D013482:Superoxide Dismutase