Optimized extraction, identification and characterization of the mosquitocidal surface layer protein from a local bacterial isolate Lysinibacillus sphaericus Q001

• Published in: Protein expression and purification 2025-03, Vol.227, p.106639, Article 106639
• Main Authors: Arshad, Hamayun, Gardner, Qurratulann Afza, Ahmad, Saira, Bukhari, Syeda Sadia, Akhtar, Muhammad
• Format: Article
• Language: English
• Subjects: Aedes - microbiology; Aedes aegypti; Animals; Bacillaceae - chemistry; Bacillaceae - genetics; Bacillaceae - isolation & purification; Bacillaceae - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - pharmacology; Insecticides - chemistry; Insecticides - isolation & purification; Insecticides - pharmacology; Larva - microbiology; Larvicidal activity; Lysinibacillus sphaericus; MALDI biotyping; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Glycoproteins - isolation & purification; Pakistan; Ribotyping; RNA, Ribosomal, 16S - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface-layer protein
• ISSN: 1046-5928; 1096-0279; 1096-0279
• DOI: 10.1016/j.pep.2024.106639

Surface layer (S-layer) is an extracellular proteinous layer consisting of two-dimensional lattice. It is typically present on archaea and also found on some bacteria. S-layer proteins from some bacteria are reported to be toxic to mosquito larvae. Here, we aimed to extract and characterize the surface layer protein from a local bacterial strain named Lysinibacillus sphaericus Q001. This bacterium was isolated from Pakistan and characterized through various biochemical tests. It was identified as Lysinibacillus sphaericus through 16S rRNA ribotyping (NCBI accession no. OQ701385.1) and matrix-assisted laser desorption/ionization (MALDI) biotyping with 2.18 ± 0.059 score. The S-layer protein was extracted by both cation exchange method and guanidinium chloride extraction method. The optimized method for the extraction and purification of S-layer yielded 35 mg of protein from 1 L culture of L. sphaericus Q001. A potential S-layer protein band (120 kDa) detected by SDS-PAGE was confirmed by bottom-up proteomics i.e., in-gel tryptic digestion of the protein followed by MALDI-TOF analysis and peptide mass fingerprinting (PMF). The insecticidal bioassays revealed that S-layer protein of L. sphaericus Q001 was toxic against Aedes aegypti larvae with LC50 value of 11 μg/ml. This shows its potential to be used as an alternative to chemical larvicides. •First report on surface layer of Lysinibacillus sphaericus Q001 from Pakistan.•Direct screening of surface layer (S-layer) protein in bacteria.•Confirmation of L. sphaericus Q001 S-layer through bottom-up proteomics.•Characterization of S-layer protein of L. sphaericus Q001.•S-layer of L. sphaericus Q001 show toxicity against Aedes aegypti larvae.