Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans.
We have studied the production of mouse tumor necrosis factor alpha (mTNF) with Streptomyces lividans as host. mTNF cDNA was fused to the alpha-amylase-encoding gene (aml) of Streptomyces venezuelae ATCC15068 at 12 amino acids (aa) downstream from the signal-peptidase cleavage site so that the aa surrounding this processing site were conserved. S. lividans containing this construct secreted mTNF at moderately high levels (1-10 micrograms/ml) as a biologically active compound of high specific activity (1 x 10(8) units/mg protein). No unprocessed pre-protein and virtually no processed protein could be detected in the cell lysates. N-terminal aa sequence analysis indicated microheterogeneity (-3 to -6 forms) at the N-terminal site of secreted mTNF. It was demonstrated that this microheterogeneity was due to aminopeptidase activity.
Laboratory of Microbiology, Rega Institute, K.U. Leuven, Belgium.
L
L
Van Mellaert
C
C
Dillen
P
P
Proost
E
E
Sablon
R
R
DeLeys
A
A
Van Broekhoven
H
H
Heremans
J
J
Van Damme
H
H
Eyssen
J
J
Anné
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002417:Cattle; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000516:alpha-Amylases / Q000235:genetics
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000516:alpha-Amylases