Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli.
Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.
Morelle
G
G
Division of Enzymtechnology, GBF-Gesellschaft für Biotechnologische Forschung, Braunschweig, F.R.G.
Frank
R
R
Meyerhans
A
A
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids
D004269:DNA, Bacterial; D010281:Parathyroid Hormone