Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus-insect cell expression system.
We report gene cloning, plasmid construction, baculovirus expression, purification, and biological activity testing of the human hematopoietic cytokine interleukin-3. cDNA was constructed from extracted total RNA of Jurkat cells. Both signal and structural fragment of interleukin-3 were cloned from this cDNA library, modified by adding a hexahistidine-tag at the C-terminus, and introduced into the pBacPAK9 transfer vector to generate recombinant baculoviruses. For protein expression High Five cells were infected either in spinner flasks or 2.5-L bioreactors in batch culture yielding levels of 1.5-3 mg L(-1) interleukin-3 in the cell culture supernatant. Interleukin-3 was purified by a single step chromatography using cobalt metal affinity resins, which yielded a highly stable and soluble protein. N-terminal amino acid sequencing of the purified interleukin-3 showed correct cleavage of the signal peptide during protein processing. The two N-glycosylation sites were found to be occupied by 100 and 35%, respectively, with an N-glycan pattern of paucimannosidic structures, which are typical for recombinant glycoproteins produced by High Five lepidopteran cells. The specific biological activity of purified interleukin-3 was several times higher when compared with different lots of commercially available material from Escherichia coli. The results indicate that the strategy we used in this experiment is a straightforward and convenient way for recombinant protein preparation and can be adapted to produce other recombinant cytokines.
Ding
Hongbiao
H
Cell Culture Technology Department, Gesellschaft für Biotechnologische Forschung GmbH (GBF), Mascheroder Weg 1, D-38124, Braunschweig, Germany.
Griesel
Carsten
C
Nimtz
Manfred
M
Conradt
Harald S
HS
Weich
Herbert A
HA
Jäger
Volker
V
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D007377:Interleukin-3 / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D019169:Jurkat Cells; D011336:Probability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D016622:Silver Staining; D021241:Spectrometry, Mass, Electrospray Ionization; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D013778:Tetrazolium Salts / Q000494:pharmacology; D014170:Transformation, Genetic / Q000235:genetics; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D007377:Interleukin-3; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D014357:Trypsin