Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.
Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.
Division of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, City of Havana, Cuba. gillian.martinez@cigb.edu.cu
Gillian
G
Martinez-Donato
Nelson
N
Acosta-Rivero
Juan
J
Morales-Grillo
Alexis
A
Musacchio
Ariel
A
Vina
Catalina
C
Alvarez
Nelvis
N
Figueroa
Ivis
I
Guerra
Jose
J
Garcia
Laura
L
Varas
Verena
V
Muzio
Santiago
S
Dueñas-Carrera
D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D018936:Hepatitis C Antigens / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D000956:Antigens, Viral; D018936:Hepatitis C Antigens; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D015678:Viral Structural Proteins; C091015:glycoprotein E2, Hepatitis C virus