Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case.

Sara Abouelasrar Salama, Mirre De Bondt, Nele Berghmans, Mieke Gouwy, Vivian Louise Soares de Oliveira, Sergio C Oliveira, Flavio A Amaral, Paul Proost, Jo Van Damme, Sofie Struyf, Mieke De Buck

Mediators of inflammation 2020

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The serum amyloid A (SAA) gene family is highly conserved and encodes acute phase proteins that are upregulated in response to inflammatory triggers. Over the years, a considerable amount of literature has been published attributing a wide range of biological effects to SAAs such as leukocyte recruitment, cytokine and chemokine expression and induction of matrix metalloproteinases. Furthermore, SAAs have also been linked to protumorigenic, proatherogenic and anti-inflammatory effects. Here, we investigated the biological effects conveyed by murine SAA3 (mu rSAA3) recombinantly expressed in Escherichia coli. We observed the upregulation of a number of chemokines including CCL2, CCL3, CXCL1, CXCL2, CXCL6 or CXCL8 following stimulation of monocytic, fibroblastoid and peritoneal cells with mu rSAA3. Furthermore, this SAA variant displayed potent in vivo recruitment of neutrophils through the activation of TLR4. However, a major problem associated with proteins derived from recombinant expression in bacteria is potential contamination with various bacterial products, such as lipopolysaccharide, lipoproteins and formylated peptides. This is of particular relevance in the case of SAA as there currently exists a discrepancy in biological activity between SAA derived from recombinant expression and that of an endogenous source, i.e. inflammatory plasma. Therefore, we subjected commercial recombinant mu rSAA3 to purification to homogeneity via reversed-phase high-performance liquid chromatography (RP-HPLC) and re-assessed its biological potential. RP-HPLC-purified mu rSAA3 did not induce chemokines and lacked in vivo neutrophil chemotactic activity, but retained the capacity to synergize with CXCL8 in the activation of neutrophils. In conclusion, experimental results obtained when using proteins recombinantly expressed in bacteria should always be interpreted with care. Copyright © 2020 Sara Abouelasrar Salama et al.

Citation

Sara Abouelasrar Salama, Mirre De Bondt, Nele Berghmans, Mieke Gouwy, Vivian Louise Soares de Oliveira, Sergio C Oliveira, Flavio A Amaral, Paul Proost, Jo Van Damme, Sofie Struyf, Mieke De Buck. Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case. Mediators of inflammation. 2020;2020:6087109