Whiteley E M, Hsu T A, Betenbaugh M J
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biotechnol Bioeng. 1997 Oct 5;56(1):106-16. doi: 10.1002/(SICI)1097-0290(19971005)56:1<106::AID-BIT12>3.0.CO;2-I.
A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired.
构建了杆状病毒-昆虫细胞系统中免疫球蛋白G（IgG）产生的模型，该模型纳入了多肽合成、寡聚体组装、蛋白质聚集和蛋白质分泌过程。此外，通过纳入文献中的体外伴侣蛋白-肽结合和解离动力学常数，考虑了伴侣蛋白保护重链和轻链多肽免于蛋白质聚集的能力。然后将模型预测结果与实验进行比较，在实验中，通过用含BiP的杆状病毒共感染昆虫细胞来共表达伴侣蛋白免疫球蛋白重链结合蛋白（BiP）。该模型预测细胞内和分泌的IgG增加近两倍，这与共表达异源IgG和BiP约3天后实验观察到的行为相似。然而，在模型模拟和实验中免疫球蛋白聚集仍然很显著，因此该模型随后被用于预测进一步提高IgG产量的策略的效果。将伴侣蛋白BiP的表达在当前实验水平上提高10倍，与无BiP时的IgG组装相比，分泌的IgG产量提高了2.5倍。或者，在杆状病毒感染周期的早期表达BiP，在不需要过量产生BiP的情况下，蛋白质分泌增加了两倍。通过改变BiP的结合和释放常数，考虑了共伴侣蛋白对BiP活性的潜在影响。利用较低的结合和释放动力学常数导致IgG分泌增加了几倍，因为多肽在更长时间内受到保护免于聚集。伴侣蛋白作用的优化策略将包括BiP-ATP构象快速的肽结合以及BiP-配体构象缓慢的肽释放。然而，即使有优化的伴侣蛋白系统，分泌动力学的限制也可能导致细胞内IgG的积累。因此，当需要增强异源蛋白的分泌时，必须考虑整个分泌途径。
