Over-expression of a human CD62L ecto-domain and a potential role of RNA pseudoknot structures in recombinant protein expression

- Optimized GS-based protein expression system for stable cell clone production.
- Increased yield of the full ectodomain of a human selectin protein, CD62L.
- Investigated effect of tertiary mRNA structures on protein expression levels.
- Pseudoknots involving start codon and signal sequences affect protein expression.

L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular region of CD62L. This resulted in excess of 20 mg/L yield of recombinant CD62L. In an attempt to understand the different expression levels among four similar CD62L constructs that differ primarily in signal sequences, we calculated the presence of potential RNA pseudoknots in their signal sequences. The results showed the presence of pseudoknots involving the start codon and between the signal sequence and gene in the mRNA of the non-expressing constructs, suggesting a potential inhibitory role of RNA pseudoknots in recombinant protein expression.