Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line.
Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.
Hains
J M
JM
Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
Aggarwal
B B
BB
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007668:Kidney; D008233:Lymphotoxin-alpha / Q000096:biosynthesis / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins