BACTERIAL EXPRESSION AND PURIFICATION OF HEPATITIS-C VIRUS CAPSID PROTEINS OF DIFFERENT SIZE

Two capsid sequences of the hepatitis C virus were cloned and expresse d in an E. coli system, One sequence (c190) comprised the complete cap sid region with 573 nucleotides. The other sequence (c125) spanned 375 5'-nucleotides lacking the hydrophobic 3'-part of the hepatitis C vir us capsid gene, A full-length and a truncated construct were chosen, s ince it is not known whether there is 3'-truncation of the hepatitis C virus capsid during protein maturation similar to the situation in so me flaviviridae. The corresponding expression clones 190/4 and 125/4 w ere constructed by polymerase chain reaction cloning into pQE-vectors. The protein expressed, pc125, which is lacking the hydrophobic carbox yterminus of the full-length capsid protein pc190, showed a stronger s ignal in western blots using anti-hepatitis C virus/EIAII-positive pat ient's serum, This could be due to better expression and/or better sol ubilization of pc125, The truncated protein pc125 displayed the predic ted molecular weight of 19 kD, whereas the full-length protein pc190 m igrated faster than expected. This could be due to intracellular prote olytic processing, giving rise to a truncated protein or to an atypica l mobility in SDS-PAGE gels caused by the hydrophobic nature of the fu ll-length protein, Both proteins were synthesized with an aminotermina l tag of six histidines that could be used for purification by Nickel chelate affinity chromatography. The elution fractions of the two prot eins showed additional bands in western blots, Most of these proteins had a mass between 2 and 16 kD and are likely to be degradation produc ts. Protein pc125 could be purified in larger quantities than pc190. U sing electroelution, a single signal was detected in western blots ind icating the purity of the hepatitis C virus capsid proteins which were obtained.