Expression and characterization of soluble human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein.
Human parainfluenza virus types 1 (hPIV-1), 2, and 3 represent significant respiratory pathogens for which no antiviral treatment is currently available. To characterize the biochemical functions of the hPIV-1 hemagglutinin-neuraminidase (HN) glycoprotein, a potential target for antiviral therapy, we cloned and expressed a soluble portion of hPIV-1 HN (amino acid residues 137-575), lacking the N-terminal hydrophobic membrane anchorage region, in insect cells using the baculovirus secretion expression system. The expressed HN protein was purified through cation-exchange chromatography followed by metal affinity chromatography, using the 6xHis epitope introduced at the carboxyl terminus of the recombinant protein. N-terminal amino acid sequence analysis of purified HN indicated that the honeybee melittin secretion signal peptide was correctly removed during post-translational processing. Further characterization revealed that the purified HN protein was N-glycosylated and exhibited neuraminidase activity whose characteristics resembled those of the native HN protein of hPIV-1 virions. The establishment of this expression and purification system has allowed us to further explore the biochemical characteristics of paramyxovirus HN and to obtain material that could be suitable for X-ray crystallography studies.
Wang
Z M
ZM
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA. michael_wang@gilead.com
Tong
L L
LL
Grant
D
D
Cihlar
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D005822:Genetic Vectors; D015707:HN Protein / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D009532:Nickel; D010222:Parainfluenza Virus 1, Human / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012965:Sodium Chloride
D015707:HN Protein; D011994:Recombinant Proteins; D012965:Sodium Chloride; D009532:Nickel; D009439:Neuraminidase