Combining gene amplification with SEAP-reporter to achieve high expression of recombinant omicron XBB.1.5 spike protein.

Most stable mammalian recombinant protein expressions use labor-intensive ELISA with target specific antibodies for screening of high-expression clones. Some alternative antibody-free reporter systems, such as the ones using intracellular green fluorescent proteins, simplified the screening of expression clones but often resulted in limited expression yields. We have previously developed a glutamine synthetase (GS)-based amplifiable gene expression system to obtain high yield expression for some difficult to express recombinant proteins. Here, we further improved the GS-based expression system by adding a secreted embryonic alkaline phosphatase (SEAP)-based reporter to allow efficient, antibody-free selection of robust recombinant expression clones. We tested the GS and SEAP combined strategy on expression of a relatively difficult protein, the soluble trimeric SARS-CoV-2 Omicron XBB.1.5 spike protein and obtained greater than 130 mg/L yield of purified protein. This represents the highest expression of coronavirus spike trimer in HEK293 cells. Thus, the combination of gene amplification with a simple, robust selection results in a high throughput and high-yield recombinant protein expression system.