[A study of protein engineering for human cardionatrin. I. Synthesis, cloning and expression of a gene analog of human atrial natriuretic polypeptide in E. coli].
An analog of the alpha-human atrial natriuretic polypeptide (alpha-hANP) gene, articulated with a peptidase inhibitor SQ20881 at its N-terminal and two prolines at the C-terminal was expressed in E. coli by cloning the reconstituted plasmid pRHL-1 in vivo. This gene analog, RH-1, comprising 154 base pairs in total, was designed to contain an equivalent of the alpha-hANP gene, capping the peptidase inhibitor SQ20881 at its 5' end with a glutamic acid codon GAA to facilitate enzymatic cleavage of the expressed end product by endoproteinase Glu-C, wedging in two proline codons CCG & CCG before the double terminal codons TGA TAG at the 3' end to retard hydrolysis of the expressed product by exopeptidase, and adding 3 restriction sites to both ends. Synthesis of the RH-1 gene was effected enzymatically by joining in predicted order the ten segments of oligodeoxynucleotides which had been chemically synthesized by the solid-phase phosphite-triester method. The synthetic gene was cloned into vector M13mp18. Phage bearing the gene analog was identified by dot blotting and restriction endonuclease mapping. Nucleotide sequence of the gene was determined by the dideoxynucleotide chain termination method.(ABSTRACT TRUNCATED AT 250 WORDS)
Ren
H
H
Institute of Basic Medical Sciences, Beijing.
D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering
D009320:Atrial Natriuretic Factor; D004247:DNA