Hitzeroth Inga I, Chabeda Aleyo, Whitehead Mark P, Graf Marcus, Rybicki Edward P
Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, South Africa.
Thermo Fisher Scientific GENEART GmbH, Regensburg, Germany.
Front Bioeng Biotechnol. 2018 Jul 16;6:101. doi: 10.3389/fbioe.2018.00101. eCollection 2018.
Human papillomaviruses (HPVs) are the causative agents of cervical cancer, the fourth most prevalent cancer in women worldwide. The major capsid protein L1 self-assembles into virus-like particles (VLPs), even in the absence of the minor L2 protein: such VLPs have successfully been used as prophylactic vaccines. There remains a need, however, to develop cheaper vaccines that protect against a wider range of HPV types. The use of all or parts of the L2 minor capsid protein can potentially address this issue, as it has sequence regions conserved across several HPV types, which can elicit a wider spectrum of cross-neutralizing antibodies. Production of HPV VLPs in plants is a viable option to reduce costs; the use of a L1/L2 chimera which has previously elicited a cross-protective immune response is an option to broaden cross-protection. The objective of this study was to investigate the effect of codon optimization and of increasing the G+C content of synthetic L1/L2 genes on protein expression in plants. Additionally, we replaced varying portions of the 5' region of the gene with the wild type () viral sequence to determine the effect of several negative regulatory elements on expression. We showed that GC-rich genes resulted in a 10-fold increase of mRNA levels and 3-fold higher accumulation of proteins. However, the highest increase of expression was achieved with a high GC-content human codon-optimized gene, which resulted in a 100-fold increase in mRNA levels and 8- to 9-fold increase in protein levels. Changing the 5' end of the gene back to its sequence decreased mRNA and protein expression. Our results suggest that the negative elements in the 5' end of are inadvertently destroyed by changing the codon usage, which enhances protein expression.
人乳头瘤病毒（HPV）是宫颈癌的致病因子，宫颈癌是全球女性中第四大常见癌症。主要衣壳蛋白L1即使在没有次要L2蛋白的情况下也能自组装成病毒样颗粒（VLP）：此类VLP已成功用作预防性疫苗。然而，仍需要开发更便宜的疫苗，以预防更广泛的HPV类型。使用全部或部分L2次要衣壳蛋白可能解决这一问题，因为它具有在几种HPV类型中保守的序列区域，可引发更广泛的交叉中和抗体。在植物中生产HPV VLP是降低成本的可行选择；使用先前引发交叉保护性免疫反应的L1/L2嵌合体是扩大交叉保护的一种选择。本研究的目的是研究密码子优化和增加合成L1/L2基因的G+C含量对植物中蛋白质表达的影响。此外，我们用野生型（）病毒序列替换了该基因5'区域的不同部分，以确定几种负调控元件对表达的影响。我们发现富含GC的基因使mRNA水平增加了10倍，蛋白质积累增加了3倍。然而，高GC含量的人密码子优化基因实现了最高的表达增加，导致mRNA水平增加了100倍，蛋白质水平增加了8至9倍。将该基因的5'端改回其序列会降低mRNA和蛋白质表达。我们的结果表明，通过改变密码子使用，该基因5'端的负元件被意外破坏，从而增强了蛋白质表达。
