Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library

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Abstract
The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tac I promoter has been used to direct the synthesis in E. coli of this SOD which is soluble, stable, and of normal specific activity. The N-terminal methionine is removed from this protein. A construction with a ribosome binding site identical to that of the lacz gene 5′ of the initiator methionine codon, resulted in low levels of SOD. An in vitro mutagenesis procedure was used to randomise the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids. Analysis of a sample of 500 clones showed that ca. 25 clones synthesised 5% or more of soluble cell, protein as SOD. The nucleotide sequences of high level expressors showed a predominance of A and T residues in the variable positions 5′ of the initiator methionine codon. An SOD mutant (ala 4 →gln) was discovered during the sequencing and shown to lack dismutation activity. Secondary structure predictions for the 5′ regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to l6s rRNA is base paired in a stem structure.