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<p>BPI, produced by polymorphonuclear leukocytes, exhibits strong antimicrobial activity against Gram-negative bacteria by binding LPS with high affinity. This study aimed to express functional recombinant human BPI (BPI53) in a prokaryotic system to evaluate its potential as a therapy against LPS-induced inflammation. The BPI53 gene was codon-optimized for prokaryotic expression and cloned into <i>E. coli</i> BL21. Colony PCR using universal pET primers confirmed successful transformation. Protein expression was induced with 1 mM IPTG, and BPI53 was purified under native conditions using a Ni–NTA affinity column. Western blotting with an anti-His-tag antibody verified recombinant protein expression. LPS binding was assessed by ELISA, while anti-inflammatory activity was evaluated in LPS-stimulated primary peritoneal macrophages by measuring pro- and anti-inflammatory cytokines (TNF-α, ILs, and NO) using commercial ELISA kits. Codon optimization increased the CAI from 0.61 to 0.81, resulted in a balanced GC content of 51.52% and enhanced mRNA stability with a ΔG value of –443.7 kcal/mol. The optimized sequence showed no tandem repeats or GC-rich regions and fewer secondary structures near the 5′ start site. The optimized BPI53 sequence was successfully expressed in <i>E. coli</i> BL21(DE3); SDS-PAGE and Western blotting confirmed the presence of a ~ 53 kDa band. ELISA demonstrated LPS binding (OD: 0.41 at 312 ng/mL; EC₅₀: 1.3 μg/mL). Functionally, 400 ng/mL BPI53 significantly reduced LPS-induced nitric oxide, IL-10, and TNF-α production, and also modulated IL-1β and IL-6 levels, indicating measurable LPS-neutralizing activity. Bioinformatic design enabled successful expression of functional human BPI53 in <i>E. coli</i>. The recombinant protein showed strong LPS-binding and anti-inflammatory activity, highlighting its potential as a cost-effective therapy for Gram-negative sepsis.&#xa0;</p>

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Bioinformatic design and expression of bactericidal permeability increasing protein (BPI) to neutralize LPS cytotoxic effects

  • 期刊论文

  • 发表日期：

    2026年2月25日

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  • Hossein Samiei-Abianeh,
  • Amirhossein Sahebkar,
  • Shahram Nazarian,
  • Amin Jalili,
  • Seyed Ali Mirhosseini

摘要

BPI, produced by polymorphonuclear leukocytes, exhibits strong antimicrobial activity against Gram-negative bacteria by binding LPS with high affinity. This study aimed to express functional recombinant human BPI (BPI53) in a prokaryotic system to evaluate its potential as a therapy against LPS-induced inflammation. The BPI53 gene was codon-optimized for prokaryotic expression and cloned into E. coli BL21. Colony PCR using universal pET primers confirmed successful transformation. Protein expression was induced with 1 mM IPTG, and BPI53 was purified under native conditions using a Ni–NTA affinity column. Western blotting with an anti-His-tag antibody verified recombinant protein expression. LPS binding was assessed by ELISA, while anti-inflammatory activity was evaluated in LPS-stimulated primary peritoneal macrophages by measuring pro- and anti-inflammatory cytokines (TNF-α, ILs, and NO) using commercial ELISA kits. Codon optimization increased the CAI from 0.61 to 0.81, resulted in a balanced GC content of 51.52% and enhanced mRNA stability with a ΔG value of –443.7 kcal/mol. The optimized sequence showed no tandem repeats or GC-rich regions and fewer secondary structures near the 5′ start site. The optimized BPI53 sequence was successfully expressed in E. coli BL21(DE3); SDS-PAGE and Western blotting confirmed the presence of a ~ 53 kDa band. ELISA demonstrated LPS binding (OD: 0.41 at 312 ng/mL; EC₅₀: 1.3 μg/mL). Functionally, 400 ng/mL BPI53 significantly reduced LPS-induced nitric oxide, IL-10, and TNF-α production, and also modulated IL-1β and IL-6 levels, indicating measurable LPS-neutralizing activity. Bioinformatic design enabled successful expression of functional human BPI53 in E. coli. The recombinant protein showed strong LPS-binding and anti-inflammatory activity, highlighting its potential as a cost-effective therapy for Gram-negative sepsis.