Palomares Laura A, López Susana, Ramírez Octavio T
Departamento de Bioingeniería, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca Morelos, México.
Biotechnol Bioeng. 2002 Jun 20;78(6):635-44. doi: 10.1002/bit.10243.
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.
在这种复杂的多聚体结构作为疫苗获得实际应用之前，病毒样颗粒的充分生产策略是必须解决的挑战之一。实现构成病毒样颗粒的蛋白质之间正确的化学计量关系，应通过使组装蛋白的浓度最大化并防止废单体的积累来提高生产力。在这项工作中，利用昆虫细胞杆状病毒表达载体系统，探索了操纵构成轮状病毒样颗粒的两种结构蛋白（VP2和VP6）之间相对浓度的策略。结果表明，感染复数是操纵表达一种或两种重组蛋白的培养物中蛋白质生产率和最大浓度的有用工具。因此，感染复数可用于提高轮状病毒样颗粒的产量。当同时产生VP2和VP6时，单独感染期间获得的VP2和VP6生产率保持不变，这表明这些生产率可用于估计共表达期间的蛋白质浓度。本文首次提出的操纵两种重组杆状病毒之间的感染时间，也被证明对控制相对蛋白质浓度有效。使用这种顺序感染构成了一种有效的生产替代方法，该方法不需要大量病毒储备且易于实施。除了VP2和VP6之外，还获得了构成轮状病毒样颗粒的其他两种蛋白质（VP4和VP7）单独生产的动力学参数。
