Scalable production and characterization of recombinant pneumococcal histidine triad protein D in Escherichia coli for vaccine development.

Streptococcus pneumoniae causes 1.5 million deaths annually, primarily in low-income countries. Pneumococcal histidine triad protein D (PhtD), a conserved 92-110 kDa surface protein, is a promising serotype-independent vaccine candidate due to its roles in adherence, zinc homeostasis, and complement evasion. This study optimized scalable production of His-tagged PhtD in Escherichia coli BL21(DE3). The PhtD gene (serotype 2, strain D39) was cloned into pET30a(+), and expression was optimized using a full factorial design of experiments, achieving up to 19.8 mg/L soluble PhtD (>90 % purity) with 0.1 mM IPTG at OD590 = 0.2 for 2 h. Lysozyme treatment (0.3 mg/mL) increased yield to 22 mg/L, a 120 % improvement over the control (10 mg/L). Purification involved sonication, ultrafiltration, and IMAC (80-100 mM imidazole wash, 200-300 mM elution), with 1 mM PMSF preventing ∼30 % yield loss from proteolysis. PhtD's identity (105-110 kDa), secondary structure (15.5 % helix, 30.4 % sheet), and thermal stability (Tm ∼102 °C) were confirmed by SDS-PAGE, Western blotting, FTIR, circular dichroism, NMR, and DSC. This optimized, statistically guided process delivers the highest reported yield of soluble full-length PhtD in cytoplasmic E. coli with documented native-like folding and exceptional stability (Tm = 102 °C), providing a robust and scalable platform for serotype-independent pneumococcal vaccine development.