Development of an improved mammalian overexpression method for human CD62L

•We previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system with 5–30mg/L yield.•We improved the GS-based method using a single-round in situ amplification to replace the multi-round gene selection and amplification.•The improved method shortens the cell line construction from 4–6month to 2months.•We also developed a MSX resistance assay as an alternative to ELISA for evaluating the expression level of stable recombinant CHO cell lines. We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5–30mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6months to produce. To shorten the construction time, we replaced the multi-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ∼5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines.