The expression of tomato prosystemin in Escherichia coli: A structural challenge.
Prosystemin is the 200-amino-acid prohormone of the 18-amino-acid polypeptide called systemin, a systemic mobile signal that activates the synthesis of defense genes in solanaceous plants in response to herbivore attacks. The unusual primary structural features of the tomato prosystemin cDNA and protein provided an extraordinary challenge in devising an expression system to obtain the full-length protein. Prosystemin expression inhibited the growth of a eukaryotic and several prokaryotic hosts used. Prosystemin was initially synthesized as a truncated protein of 185 amino acids in length using a T7 RNA polymerase expression system in E. coli strain BL21[DE3]. The truncation was found to be due to two factors: (1) the intramolecular associations of the 5' coding region of the prosystemin sequence with the expression vector's ribosome binding site and (2) the presence of a translation start site just prior to the amino acid methionine at position 15. Mutations that permitted the synthesis of the full-length prosystemin protein were introduced into the amino-terminal 5' coding region of the prosystemin cDNA. A 199-amino-acid recombinant prosystemin lacking the N-terminal methionine was purified from lysates and confirmed by N-terminal amino acid sequence and immunoblot analysis.
Délano
J P
JP
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Dombrowski
J E
JE
Ryan
C A
CA
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D017343:Genes, Plant; D005822:Genetic Vectors; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D011994:Recombinant Proteins; C073705:prosystemin protein, Lycopersicon esculentum