Heterologous expression and purification of the infectious salmon anemia virus hemagglutinin esterase.
This study presents the heterologous production and purification of a soluble and functional form of the hemagglutinin esterase (HE) of the infectious salmon anemia virus (ISAV) isolate 4 (Glesvaer/2/90). The HE possesses receptor binding and receptor destroying enzyme (RDE) activity and is probably involved in the infection process. The recombinant HE protein (recHE 4) was expressed in insect cells (Sf9) using the baculovirus expression vector system. Both the transmembrane region and the cytoplasmic tail were deleted, and a C-terminal His(6)-tag was attached to facilitate identification and purification of the recHE 4 protein. As determined by Western analysis the recHE 4 was secreted at 20 degrees C and not at 28 degrees C. By testing three HE constructs differing in their promoter and secretion signal sequences it was clear that the HE's own secretion signal sequence is more important than the promoter with respect to the amount of secreted recHE 4 obtained under the conditions used. A one-step purification by nickel-affinity chromatography resulted in a highly purified recHE 4, identified by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Also, the recHE 4 is glycosylated and contains disulfide bridges within the molecule. Functional studies including the verification of the receptor destroying enzyme (RDE) activity as well as the binding to Atlantic salmon erythrocytes (hemagglutination) indicate that the recHE 4 has similar functions as its native counterpart. In conclusion, insect cells secrete a functional form of the ISAV 4 HE. This is suitable for further analyses on its function and immunogenicity.
Müller
Anita
A
Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Tromsø, Norway. anitamu@nfh.uit.no
Solem
Stein Tore
ST
Karlsen
Christian R
CR
Jørgensen
Trond Ø
TØ
D000115:Acetylesterase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D004795:Enzyme Stability; D004912:Erythrocytes / Q000166:cytology; D006031:Glycosylation; D006384:Hemagglutination; D006389:Hemagglutinins, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D007313:Insecta; D046829:Isavirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D020317:Salmo salar / Q000821:virology; D014760:Viral Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D006389:Hemagglutinins, Viral; D014760:Viral Fusion Proteins; C060814:hemagglutinin esterase; D000115:Acetylesterase