Glycosylation and high-level secretion of human tumour necrosis factor-beta in recombinant baculovirus-infected insect cells.
Human tumour necrosis factor-beta (TNF-beta) was produced in eukaryotic cells using the insect baculovirus cloning and expression system. A novel insect signal sequence, the honey-bee (Apis mellifera) prepromelittin secretory sequence, was used to aid in the post-translational modifications, glycosylation and secretion of recombinant human TNF-beta. Human TNF-beta cDNA was cloned using the insect baculovirus vector pAcC4s. Expression of the human TNF-beta was regulated by the insect Autographa californica nuclear-polyhedrosis-virus polyhedrin promoter. The 5' end of the TNF-beta cDNA was fused to the honey-bee prepromelittin signal sequence on the baculovirus vector. Insect [Spodoptera frugiperda (Sf9)] cells infected with the recombinant baculovirus secreted high levels of recombinant human TNF-beta into the culture medium. The amount of TNF-beta secreted by the Sf9 cells was estimated to be 28 micrograms of TNF-beta/ml of culture medium at 60-72 h post infection. The secreted human TNF-beta was a 22.5 kDa polypeptide which was glycosylated. Amino acid sequencing of the N-terminus of the recombinant human TNF-beta purified from the infected Sf9-cell culture confirmed that the secreted product was indeed human TNF-beta. This demonstrates that the honey-bee prepromelittin signal sequence was efficiently recognized and accurately cleaved in the Sf9 insect cells. The insect-derived TNF-beta exhibited a high cytotoxic activity similar to that of the native human TNF-beta when assessed by cytotoxic assays using murine L929 cells. Thus the insect baculovirus expression vector can be used for the production of abundant quantities of biologically active, glycosylated human TNF-beta protein.
Chai
H
H
Department of Chemical Engineering, Faculty of Engineering, National University of Singapore.
Vasudevan
S G
SG
Porter
A G
AG
Chua
K L
KL
Oh
S
S
Yap
M
M
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D017421:Sequence Analysis
D002241:Carbohydrates; D018076:DNA, Complementary; D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins