Bacterial expression and purification of hepatitis C virus capsid proteins of different size.
Two capsid sequences of the hepatitis C virus were cloned and expressed in an E. coli system. One sequence (c190) comprised the complete capsid region with 573 nucleotides. The other sequence (c125) spanned 375 5'-nucleotides lacking the hydrophobic 3'-part of the hepatitis C virus capsid gene. A full-length and a truncated construct were chosen, since it is not known whether there is 3'-truncation of the hepatitis C virus capsid during protein maturation similar to the situation in some flaviviridae. The corresponding expression clones 190/4 and 125/4 were constructed by polymerase chain reaction cloning into pQE-vectors. The protein expressed, pc125, which is lacking the hydrophobic carboxyterminus of the full-length capsid protein pc190, showed a stronger signal in western blots using anti-hepatitis C virus/EIAII-positive patient's serum. This could be due to better expression and/or better solubilization of pc125. The truncated protein pc125 displayed the predicted molecular weight of 19 kD, whereas the full-length protein pc190 migrated faster than expected. This could be due to intracellular proteolytic processing, giving rise to a truncated protein or to an atypical mobility in SDS-PAGE gels caused by the hydrophobic nature of the full-length protein. Both proteins were synthesized with an aminoterminal tag of six histidines that could be used for purification by Nickel chelate affinity chromatography. The elution fractions of the two proteins showed additional bands in western blots. Most of these proteins had a mass between 2 and 16 kD and are likely to be degradation products. Protein pc125 could be purified in larger quantities than pc190.(ABSTRACT TRUNCATED AT 250 WORDS)
Handschuh
G
G
Department of Virus Research, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Caselmann
W H
WH
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D002614:Chelating Agents; D002846:Chromatography, Affinity; D003201:Computers; D004563:Electrochemistry / Q000379:methods; D004926:Escherichia coli / Q000502:physiology*; D015870:Gene Expression; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008670:Metals; D008969:Molecular Sequence Data; D017421:Sequence Analysis
D002614:Chelating Agents; D008670:Metals