Overproduction of soluble, extracellular cytotoxin alpha-sarcin in Escherichia coli.
The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.
Parente
D
D
Menarini Ricerche S.p.A., Department of Biotechnology, Pomezia, Rome, Italy.
Raucci
G
G
D'Alatri
L
L
d'Estais
G
G
Novelli
S
S
Pacilli
A
A
Saccinto
M P
MP
Mele
A
A
De Santis
R
R
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003603:Cytotoxins / Q000096:biosynthesis* / Q000302:isolation & purification; D004722:Endoribonucleases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D005656:Fungal Proteins; D005822:Genetic Vectors; D018796:Immunoconjugates / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis
D003603:Cytotoxins; D005656:Fungal Proteins; D018796:Immunoconjugates; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C014801:alpha-sarcin; D004722:Endoribonucleases