Fallahee Ian, Hawiger Daniel
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA.
Antibodies (Basel). 2024 Mar 11;13(1):18. doi: 10.3390/antib13010018.
There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody-drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein-split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody-siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications.
人们对重组蛋白的生产，尤其是用于研究和治疗的双特异性抗体及抗体药物偶联物有着浓厚的兴趣。在此，我们展示了一种高度通用的质粒系统，该系统通过转染DNA的游离保留实现了稳定的Expi293细胞池的快速生成。通过一个弱化的内部核糖体进入位点将蛋白质表达与嘌呤霉素抗性联系起来，我们获得了产生感兴趣蛋白质的稳定细胞池。此外，分裂内含肽-分裂嘌呤霉素介导的两个独立蛋白质表达盒的选择，使得能够稳定生产双特异性抗体样分子或具有不同C末端重链修饰的抗体，例如一条链上带有抗原而另一条链上带有分选酶标签。我们还使用这个新型表达系统生成了分泌分选酶A Δ59变体Srt4M的稳定Expi293细胞池。利用这些试剂，我们制备了位点特异性药物与抗体比例为1的抗体-siRNA偶联物。我们预计本文所述的简单、稳健且快速的稳定蛋白质表达系统将适用于多种应用。
