Abstract:
SPRASA (Sperm protein reactive with anti-sperm antibody), with a role in fertility, was independently identified by two groups in the earlier part of this century. This protein has high structural and sequence similarity to lysozyme c and has been classified as a lysozyme-like protein, within the family of c-type lysozymes. Research has identified a role for SPRASA in fertility, in the fertilisation process and SPRASA has also been identified as a cancer testis antigen. However, many aspects of SPRASA’s role in biology remains elusive, almost nothing is known about its molecular function. Correctly folded protein is required to investigate the molecular function of hSPRASA. Bacterial expression, while popular for recombinant protein expression, results in hSPRASA being expressed within inclusion bodies. The research set out to: (A) express folded hSPRASA in Escherichia coli and in Freestyle™ Human embryonic kidney (HEK293F) cells (B) crystallise and investigate the glycan binding ability of hSPRASA. Rapid dilution, on-column refolding, buffer exchange into acidic conditions prior to dilution into low additive refolding conditions were investigated in refolding hSPRASA. These methods largely resulted in the aggregation of hSPRASA. Refolding was more complex than previously thought despite structural similarities to lysozyme (refolding model). However, the use of refolding screens resulted in promising refolding conditions being identified. Refolding conditions containing low concentrations of Arginine (aggregation suppressor) were among those identified, requiring further investigation. Successful expression of hSPRASA in HEK293F cells was confirmed using both anti-SPRASA antibodies in a Western Blot and peptide fingerprinting using mass spectrometry. From this, the next stage would be upscaling hSPRASA expression in HEK293F cells to produce sufficient yields for crystallisation and glycoarray experiments.
