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Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Antibacterial activity of recombinant hCAP18/LL37 protein secreted from Pichia pastoris
Soon-ja Kim1, Renshu Quan, Sung-Jin Lee
1Department of Biochemistry, College of Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea. hankyol2@hanmail.net
Abstract:
Human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) was expressed in Pichia pastoris and its antibacterial activity was tested against pathogenic bacteria. The full length ORF of hCAP18/LL37 was cloned into the pPICZaA vector followed by integration into the genomic AOX1 gene of P. pastoris. Agar diffusion assay demonstrated that the different hCAP18/LL37 transformants showed various antibacterial activities against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis. The secreted form of hCAP18/LL37 exhibited its maximum activity after 72 h incubation with 2% methanol in MM media, not in BMM. This result suggests that the yeast secreted expression system can be used as a production tool of antimicrobial peptides for industrial or pharmaceutical application.
Insights
The yeast Pichia pastoris successfully produced human antimicrobial peptide hCAP18/LL37, showing antibacterial activity against common pathogens. This demonstrates a viable system for producing antimicrobial peptides for pharmaceutical use.
Area of Science:
- Biotechnology
- Microbiology
- Molecular Biology
Background:
- Antimicrobial peptides (AMPs) are crucial for innate immunity.
- The human cathelicidin antimicrobial peptide CAP18/LL37 (hCAP18/LL37) possesses broad-spectrum antibacterial activity.
- Developing efficient production systems for AMPs is vital for therapeutic applications.
Purpose of the Study:
- To express the human antimicrobial peptide CAP18/LL37 (hCAP18/LL37) in the yeast Pichia pastoris.
- To evaluate the antibacterial activity of the secreted hCAP18/LL37 against pathogenic bacteria.
- To assess the potential of Pichia pastoris as a production platform for antimicrobial peptides.
Main Methods:
- Cloning the full-length hCAP18/LL37 open reading frame (ORF) into the pPICZaA vector.
- Integrating the vector into the AOX1 gene of Pichia pastoris.
- Assessing antibacterial activity using agar diffusion assays against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis.
Main Results:
- Pichia pastoris transformants expressing hCAP18/LL37 exhibited varying degrees of antibacterial activity.
- The secreted hCAP18/LL37 showed maximum activity after 72 hours of incubation in MM media with 2% methanol.
- Antibacterial activity was observed against Staphylococcus aureus, Micrococcus luteus, and Salmonella gastroenteritis.
Conclusions:
- Pichia pastoris is a suitable host for the secreted expression of human antimicrobial peptide hCAP18/LL37.
- The study validates a yeast-based expression system for producing antimicrobial peptides.
- This system holds potential for industrial and pharmaceutical applications in antimicrobial peptide production.
