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.

中文翻译：

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2020年被细菌产品污染的商业重组表达免疫调节蛋白的生物学特性：SAA3案例。

血清淀粉样蛋白A（SAA）基因家族是高度保守的，并编码响应炎症触发而上调的急性期蛋白。多年来，已经发表了大量文献，将广泛的生物学效应归因于SAA，例如白细胞募集，细胞因子和趋化因子表达以及基质金属蛋白酶的诱导。此外，SAA还与致瘤，促动脉粥样硬化和抗炎作用有关。在这里，我们调查了在大肠杆菌中重组表达的鼠SAA3（mu rSAA3）传达的生物效应。我们用mu rSAA3刺激了单核细胞，成纤维细胞和腹膜细胞后，观察到许多趋化因子的上调，包括CCL2，CCL3，CXCL1，CXCL2，CXCL6或CXCL8。此外，这种SAA变体在体内显示出强大的功能通过激活TLR4募集嗜中性粒细胞。然而，与在细菌中重组表达衍生的蛋白质有关的主要问题是各种细菌产物如脂多糖，脂蛋白和甲酰化肽的潜在污染。这在SAA的情况下尤其重要，因为目前在重组表达衍生的SAA与内源性来源（即炎症血浆）的SAA之间存在生物学活性差异。因此，我们通过反相高效液相色谱（RP-HPLC）对商品重组mu rSAA3进行了纯化，使其同质化，并重新评估了其生物学潜力。RP-HPLC纯化的mu rSAA3不能诱导趋化因子并且体内缺乏中性粒细胞趋化活性，但在中性粒细胞活化中保留了与CXCL8协同作用的能力。总之，使用细菌中重组表达的蛋白质时获得的实验结果应始终谨慎解释。