Strategies for manipulating the relative concentration of recombinant rotavirus structural proteins during simultaneous production by insect cells.
Adequate production strategies of virus-like particles are among the challenges that must be addressed before such complex multimeric structures find practical applications as vaccines. Attainment of the correct stoichiometric relation between proteins that constitute virus-like particles should result in an increased productivity by maximizing the concentration of assembled proteins and preventing the accumulation of waste monomers. In this work, strategies for manipulating the relative concentration between two of the structural proteins that constitute rotavirus-like particles (VP2 and VP6) were explored using the insect cell baculovirus expression vector system. It was shown that multiplicity of infection is a useful tool for manipulating protein production rates and maximum concentrations in cultures expressing one or two recombinant proteins. Thus, multiplicity of infection can be employed for improving production of rotavirus-like particles. VP2 and VP6 production rates obtained during individual infections remained unchanged when both were simultaneously produced, indicating that such rates can be utilized for estimating protein concentrations during coexpression. Manipulation of the time of infection between the two recombinant baculoviruses, proposed here for the first time, also proved to be effective for controlling the relative protein concentrations. The use of such sequential infections constituted an effective production alternative that does not require high amounts of virus stocks and is easy to implement. In addition to VP2 and VP6, kinetic parameters for the individual production of the other two proteins (VP4 and VP7) that constitute rotavirus-like particles were also obtained.
Palomares
Laura A
LA
Departamento de Bioingeniería, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca Morelos, México. tonatiuh@ibt.unam.mx
López
Susana
S
Ramírez
Octavio T
OT
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D005822:Genetic Vectors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism*; D018411:Spodoptera; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000378:metabolism*
D011994:Recombinant Proteins; D015678:Viral Structural Proteins