Molecular cloning, expression and purification of protein 2A of hepatitis A virus.
Expression of the protein 2A of Hepatitis A virus (HAV), spanning amino acids 764 through 981 of the viral polyprotein results in a strong inhibition of cap-dependent translation (Maltese et al., 2000). However, the molecular mechanism responsible has remained unclear, in part because the HAV 2A protein was not available in amounts large enough to allow biological or structural studies. To address this issue, a cDNA representation of the sequences encoding HAV 2A was generated by PCR, using primers that introduced an AUG triplet, and a sequence coding for 6 histidine residues at the 5'- and 3'-termini of the genomic sequence, respectively. The cDNA fragment was introduced by cassette exchange in the inducible expression vector pQE-60, and the construct was propagated in bacteria E. coli M15 which constitutively expresses the lac repressor. Upon induction with IPTG (1 mM), HAV 2A was visualized by SDS-PAGE of bacterial lysates as a prominent band M(r) = 21 kDa. The identity of the polypeptide was confirmed by both MALDI-TOF peptide mapping and direct amino acid sequencing. The His-tagged HAV 2A was extracted from bacterial pellets under totally denaturing conditions (6 M urea), subjected to Ni(++)-Sepharose affinity chromatography, allowed to refold while still attached to the matrix, and eluted with 250 mM Imidazole. Contaminant material was partly removed by differential ammonium sulfate precipitation. The protein was further concentrated (Vivaspin centrifugal concentrator), the insoluble material (if present) was discarded, and the homogeneity of the dispersion was ascertained by light scattering. SDS-PAGE revealed that in addition to the main protein (Mr = 21 kDa), a second one of apparent Mr = 14 kDa was always present in variable amounts. The proportion of the latter tended to increase with aging of the preparation. Edman degradation analysis proved that the 14 kDa protein resulted from the cleavage of HAV 2A at a so far undetected scissile bond Gly856/Val857 of the viral polyprotein. A first attempt to crystallize the protein by the hanging drop procedure yielded only small crystals containing exclusively the 14 kDa derivative of HAV 2A. Western blot analysis of HeLa cell extracts that had been incubated with the His-tagged HAV 2A so purified failed to reveal any change in the electrophoretic mobility of the eukaryotic initiation factor (eIF) 4G I.
Di Napoli
A
A
Laboratoire de Virologie Moléculaire et Structurale UMR 2472 CNRS-INRA, Gif-sur-Yvette, France.
Maltese
E
E
Bucci
M
M
Pagnotti
P
P
Seipelt
J
J
Duquerroy
S
S
Pérez Bercoff
R
R
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015967:Gene Expression Regulation, Viral; D030041:Hepatitis A virus / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins
D014764:Viral Proteins; D006639:Histidine; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus