27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity.
In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.
Nishikawa
S
S
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Matsuo
N
N
Isaka
Y
Y
Uesugi
S
S
Miura
K
K
Ohtsuka
E
E
Takeda
K
K
Konno
K
K
Ikehara
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D006801:Humans; D008233:Lymphotoxin-alpha / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013329:Structure-Activity Relationship
D008233:Lymphotoxin-alpha; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins