Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.
The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.
Johansson
T
T
Abo Akademi University, Department of Biochemistry and Pharmacy, Turku, Finland.
Enestam
A
A
Kronqvist
R
R
Schmidt
M
M
Tuominen
N
N
Weiss
S A
SA
Oker-Blom
C
C
D000818:Animals; D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D006031:Glycosylation; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012412:Rubella virus / Q000235:genetics*; D018411:Spodoptera; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins