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Platelet factor 4 is a cytokine released into the bloodstream by activated platelets where it plays a pivotal role in etiology and diagnosis of heparin-induced thrombocytopenia. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures. To this end, a three-inone primary construct was designed from which three secondary constructs can be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable chemical factors including IPTG, Triton X-100, sucrose, and glycine were individually investigated at the outset. In the next step, according to a fractional factorial approach, the synergistic effects of IPTG, Triton X-100, and glycine on secretion were further investigated. To ascertain the structure and function of the secreted recombinant proteins, dynamic light scattering was utilized to confirm the rPF4 tetramerization and heparinmediated ultra-large complex formation. Moreover, Raman spectroscopy and Western blotting were exploited to evaluate the secondary and quaternary structures, respectively. The type II secretory pathway was proven to be superior to type I in the case of rPF4 secretion. Supplementation with chemical enhancers improved the protein secretion mediated by the Type II system to approximately more than 500 μg/mL. Large quantities of native rPF4 up to 20 mg were purified as the culture medium was scaled up to 40 mL. Western blotting confirmed the formation of dimers and tetramers in the secreted rPF4 proteins. Dynamic light scattering revealed the rPF4 oligomerization into of larger complexes of approximately 100-1200 nm in size following heparin supplementation, implying proper protein folding and tetramerization. Moreover, the rPF4 secondary structure was found to be 43.5% Random coil, 32.5% β-sheet, 18.6% α-helix and 4.9% Turn, which is in perfect agreement with the native structure. Our results indicate that the gram-negative type II bacterial secretory system holds a great promise as a reliable protein production strategy with industrial applications.

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Background: Platelet factor 4 is a cytokine released into the bloodstream by activated platelets and plays a pivotal role in heparin-induced thrombocytopenia etiology and diagnosis. Therefore, a sustainable source of recombinant PF4 with structural and functional similarity to its native form is urgently needed to be used in diagnostic procedures. To this end, a three-in-one primary construct was designed and custom synthesized based on the pET26b backbone from which three secondary constructs could be derived each capable of employing either type I, type II secretory or cytoplasmic pathways. Protein expression and secretion were performed in Escherichia coli BL-21 (DE3) and were confirmed by SDS-PAGE and Western blotting. To further enhance protein secretion, the effect of several controllable factors including IPTG, Triton X-100, Sucrose, and Glycine were individually investigated at first. In the next step, according to fractional factorial approach, the synergistic effect of IPTG, Triton X-100, and Glycine on secretion was further investigated. To ascertain the structure and function of the secreted recombinant proteins, Dynamic light scattering was utilized and confirmed rPF4 tetramerization and heparin-mediated ultra-large complex formation. Moreover, Raman spectroscopy was exploited to determine the rPF4 secondary structure. Results: Type II secretory pathway was proven to be superior over type I in the case of rPF4 secretion into the extracellular milieu. Protein secretion mediated by Type II was enhanced to approximately more than 700 μg/ml. Large quantities of native rPF4 up to 20 mg was purified upon a minor scale up to 40 ml of culture medium. Dynamic light scattering unveiled native rPF4 quaternary structure revealing the formation of tetramers having an average size of 10 nm and formation of larger complexes of approximately 100-1200 nm in size following heparin supplementation, implying proper protein folding, tetramerization, and antigenicity. Analysis of the Zeta potential on approximately 600 μg/ml of rPF4 revealed a 98 mV positive charge which further confirms protein folding. Moreover, rPF4 secondary structure was determined to be 43.5% Random coil, 32.5% β-sheet, 18.6 % α-helix and 4.9 % Turn, which is in perfect agreement with the native structure. Conclusion: our results indicate that the gram-negative type II bacterial secretory system holds a great promise to be employed as a reliable protein production strategy with favorable industrial application. However, further efforts are required to realize the full potential of secretory pathways regarding their application to proteins with distinct characteristics. Key words: Platelet Factor 4, Recombinant Protein Secretion, Type I Protein Secretion, Type II Protein Secretion