Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahagno, Yuseong-gu, Daejeon 305-701, Republic of Korea.
J Biosci Bioeng. 2013 Aug;116(2):246-52. doi: 10.1016/j.jbiosc.2013.02.005. Epub 2013 Mar 13.
Owing to simple mechanism of linking and efficient display of proteins, plasmid display system in which proteins are physically linked to plasmids, has been considered as a promising emerging tool in protein engineering. We used human Oct-1 DNA-binding domain (DBD) which can bind to octameric DNA sequence (5'-ATGCAAAT-3') with high affinity, as a potential anchoring motif for plasmid display system. Using three model proteins, histidine hexamer (His6), glutathione S-transferase (GST) and antibody fragment, we confirmed that Oct-1 DBD fused proteins were strongly linked to plasmids and their linking were conserved for entire process of in vitro selection. Also, the feasibility of this display system was examined using several enrichment experiments from binary libraries. Using Oct-1 plasmid display system, the GST-displayed plasmids were successfully enriched 8500-fold from a large excess (10⁴ fold) of negatives (non-GST plasmid). From the results, Oct-1 DBD-based plasmid display system allows the rapid and facile in vitro selection and can be a useful tool in discovering functional proteins from large libraries.
由于连接机制简单且能够高效地展示蛋白质，因此质粒展示系统（其中蛋白质与质粒通过物理方式相连接）被认为是一种很有前途的新兴蛋白质工程工具。我们使用了与人 Oct-1 DNA 结合域（DBD），该结构域可以与八聚体 DNA 序列（5'-ATGCAAAT-3'）高亲和力结合，将其作为质粒展示系统的潜在锚定基序。通过使用三种模型蛋白（组氨酸六聚体（His6）、谷胱甘肽 S-转移酶（GST）和抗体片段），我们证实了融合有 Oct-1 DBD 的蛋白能够与质粒紧密结合，并且其连接在体外选择的整个过程中是保守的。此外，我们还通过几种二元文库的富集实验来检验该展示系统的可行性。使用 Oct-1 质粒展示系统，我们能够从大量过剩（10⁴ 倍）的阴性（非 GST 质粒）中成功地将 GST 展示质粒富集 8500 倍。结果表明，基于 Oct-1 DBD 的质粒展示系统可以实现快速、简便的体外选择，并且可以成为从大型文库中发现功能蛋白的有用工具。
