Beta-subunit of human chorionic gonadotropin hormone and firefly luciferase simultaneously synthesized in insect cells using a recombinant baculovirus are differentially expressed and transported.
A recombinant baculovirus vAc beta hCG-luc was constructed that carried the cDNAs encoding firefly luciferase (luc) and beta-subunit of human chorionic gonadotropin (beta hCG) placed under the transcriptional control of individual copies of the baculovirus polyhedrin gene promoter. The simple, rapid, and sensitive detection of LUC expression was used for selecting recombinant viruses that simultaneously expressed beta hCG, which was identical in all respects to that synthesized using a recombinant baculovirus carrying the beta hCG gene alone. Immunofluorescence staining of virus-infected cells using anti-LUC antibodies revealed that LUC, a nonglycosylated, intracellular protein was retained within the cells whereas beta hCG, an extensively glycosylated, secretory protein, was processed and secreted into the culture medium. LUC and beta hCG were both immunoreactive on Western blot. beta hCG was bioactive, as evident from its ability to associate with alpha hCG and bind with the receptor and produce testosterone in an in vitro mouse Leydig cell assay system. Comparison of recombinant LUC and beta hCG synthesized by the virus-infected insect cells surprisingly revealed that the level of the former was quantitatively higher by at least 10-fold than the latter. A blot of total RNA isolated from vAc beta hCG-luc-infected insect cells, when probed with probes corresponding to the 3' region of the beta hCG or luc genes, indicated differential transcription of the two genes. Computer-aided sequence analysis indicated extensive secondary structure and stem-loop complex-forming potential of the beta hCG gene, which could be responsible for the transcriptional difference observed.
Hasnain
S E
SE
Eucaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Nakhai
B
B
Ehtesham
N Z
NZ
Sridhar
P
P
Ranjan
A
A
Talwar
G P
GP
Jha
P K
PK
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D018997:Chorionic Gonadotropin, beta Subunit, Human; D001517:Coleoptera / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects / Q000502:physiology; D008156:Luciferases / Q000096:biosynthesis* / Q000378:metabolism; D008297:Male; D051379:Mice; D009036:Moths; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D011974:Receptors, LH / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013816:Thermodynamics; D014158:Transcription, Genetic; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D012333:RNA, Messenger; D011974:Receptors, LH; D011994:Recombinant Proteins; D008156:Luciferases