Production of poly(L-aspartyl-L-phenylalanine) in Escherichia coli.
The dipeptide, Asp-Phe, is the mother compound of a sweetener, aspartame (L-aspartyl-L-phenylalanine methyl ester). Two 12-mer partially complementary nucleotides that are designed, if expressed correctly, to code for Asp-Phe-Asp-Phe were synthesized to yield long and double-stranded DNA by annealing and ligation. After addition of two stop codons to the polymeric DNA, the DNA segment was inserted between the Escherichia coli trp promoter and a transcriptional terminator derived from phage fd, resulting in plasmid pDF60 that would express a protein, (1Met-10Ile)-(Asp-Phe)61. Another plasmid, pCD111, that would express a fused protein with a constitution of the NH2-terminal 167 amino acids mostly from prochymosin and the following (Asp-Phe)61, was also constructed. The polymeric DNA on pDF60 was very unstable in most of E. coli recA strains, except for strain JM109. Upon induction of the trp promoter with beta-indoleacrylic acid, E. coli JM109 harboring pDF60 formed inclusion bodies which were observed under an optical microscope. However, analyses of the inclusion bodies revealed that they consisted of many species of proteins derived from the host strain with a small amount of the poly(Asp-Phe), which could be detected by an immunological method with anti-poly(Asp-Phe) antibody. On the other hand, pCD111 directed the synthesis of the fused poly(Asp-Phe) as inclusion bodies, which was calculated to be 11.2% of the total cellular proteins.
Murata
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Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Horinouchi
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Beppu
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D000595:Amino Acid Sequence; D001483:Base Sequence; D012085:Chymosin / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
D004274:DNA, Recombinant; D004792:Enzyme Precursors; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011108:Polymers; D011993:Recombinant Fusion Proteins; C027751:poly(L-aspartyl-L-phenylalanine); C032443:prorennin; D012085:Chymosin