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

Abstract

The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tacI 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 t e variable positions 5' of the initiator methionine codon. An SOD mutant (ala +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 16s rRNA is base paired in a stem structure.

Design of Novel Conformational and Genotype-Specific Antigens for Improving Sensitivity of Immunoassays for Hepatitis C Virus-Specific Antibodies

“…The MEFA 7.1 and 7.2. constructs were genetically engineered for expression in Saccharomyces cerevisiae under the regulation of the ADH2/GAPDH hybrid promoter. The MEFA 7.1 and MEFA 7.2 antigens are chimeric polypeptides consisting of recombinant human superoxide dismutase (rhSOD), which is used to increase the level of expression of the recombinant proteins in yeast (15,16), and different epitopes of the HCV genome (see Fig. 1B).…”

Th1-like dominance in high-risk first-degree relatives of Type I diabetic patients

“…Primer sequences for the internal control, Cu,ZnSOD and for cytokine-specific primer sequences for interleukin-4 (IL-4) and IFN-g have been described previously [12]. The predicted sizes of the single PCR products are: Cu,ZnSOD, 393 bp [24]; IL-4, 176 bp [25] and IFN-g, 129 bp [26] according to the hu-man cDNA. Interferon gamma and IL-4 primers were designed as described previously [27] in such a way that they anneal to sequences located in different exons, thus making it possible to distinguish by size, amplified cDNA from amplified contaminating genomic DNA.…”

Superoxide Dismutase from the Silkworm,Bombyx mori: Sequence, Distribution, and Overexpression

“…3,4,13) cDNAs encoding SODs from diverse organisms have been cloned, some of which were overexpressed in various hosts. [14][15][16][17][18][19][20][21] In spite of its important role in the regulation of ROS, the cDNA encoding a SOD of the silkworm is not yet available.…”