Expression and structural properties of a chimeric protein based on the ectodomains of E1 and E2 hepatitis C virus envelope glycoproteins

Abstract

1Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which 2 are involved in viral attachment and entry into target cells. We have obtained in 3 insect cells infected by recombinant baculovirus a chimeric secreted recombinant 4 protein, E1 341 E2 661, containing the ectodomains of E1 and E2. The described 5 procedure allows the purification of approximately 2 mg of protein from 1 L of 6 culture media. Sedimentation velocity experiments and SDS-PAGE in the absence 7 of reducing agents indicate that the protein has a high tendency to self-associate, the 8 dimer being the main species observed. All the oligomeric forms observed maintain 9 a conformation which is recognized by the conformation-dependent monoclonal 10 antibody H53 directed against the E2 ectodomain. The spectroscopic properties of 11 E1 341 E2 661 are those of a three-dimensionally structured protein. Moreover, the 12 chimeric protein is able to bind to human antibodies present in HCV-positive human 13 sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to 14 study the structure-function relationship of HCV envelope proteins. 15 16

High-yield production of a chimeric glycoprotein based on permuted E1 and E2 HCV envelope ectodomains

“…Following this procedure, approximately 7-8 mg of E2 661 E1 340 protein were obtained from 1 L of culture media. This value represents 4-fold the yield obtained for E1 341 E2 661 (2 mg per liter) (Tello et al, 2010).…”

“…In order to connect E2 and E1 ectodomains by a flexible, protease susceptible peptide, the E1 gene was subcloned into the pProEx-HTb vector (Life Technologies, Grand Island, NY, USA), which contains the sequence encoding the TEV protease recognition site upstream of the cloning site. For this subcloning, E1 gene was amplified by PCR using the pAcGP67A-E1 340 -E2 661 (Tello et al, 2010) vector as template and the primers cg cc atg gaa ttc atg TAC CAA GTG CGC AAC (forward, Nco I restriction site underlined) and ca gcggccgc tca GAT CCG GAG CAG CTG (reverse, Not I site underlined). The resulting plasmid, pPROEX-E1 340 was used as template for a second PCR reaction using the same E1 reverse primer and the following forward primer: ga aga tct gat tac gat atc cca cg, thus obtaining a fragment containing spacer-TEV protease sequence preceeding E1, as well as a Bgl II restriction site (underlined).…”

“…E2 661 E1 340 was immunoprecipitated with H53 monoclonal antibody following the procedure previously used for E1 341 E2 661 (Tello et al, 2010) . The MAb H53 is conformation-dependent and was a generous gift of Dr. Jean Dubuisson.…”

“…Cells were grown and protocols were carried out as described in Materials and methods. As described for E1 341 E2 661 (Tello et al, 2010), the protein was expressed in a soluble form and secreted to the extracellular medium.…”

“…In order to overcome this problem, a recombinant chimeric protein containing both E1 and E2 ectodomains connected by a small hydrophilic peptide, E1 341 E2 661 , has been previously shown to have all the features of a correctly folded polypeptide (Tello et al, 2010). In this work, the cloning, expression and purification of a new chimeric protein based on permuted E1 and E2 ectodomains in a baculovirus/insect cell system is described.…”