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Updated: Mar 13, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia
De-Mei Meng1, Jing-Fang Zhao1, Xiao Ling1
1China International Science and Technology Cooperation Base of Food Nutrition/Safety and Medicinal Chemistry, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
Abstract:
The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 μg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 μg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.
Insights
Recombinant PaDef peptide from avocado was successfully expressed in Pichia pastoris. This peptide shows broad-spectrum antimicrobial activity, offering potential for new antibacterial drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- The antimicrobial peptide PaDef, isolated from Mexican avocado fruit, inhibits growth of E. coli and S. aureus.
- Previous research established PaDef's antimicrobial properties.
Purpose of the Study:
- To express and purify a recombinant N-terminal 6 × His tagged PaDef (rPaDef) in Pichia pastoris.
- To determine optimal conditions for rPaDef secretion and evaluate its antimicrobial activity.
Main Methods:
- Heterologous expression of rPaDef in Pichia pastoris.
- Optimization of culture conditions (methanol concentration, incubation time, temperature, pH).
- Purification using one-step nickel affinity chromatography and antimicrobial activity assays.
Main Results:
- Optimal rPaDef expression achieved at 1.5% methanol, 28°C, pH 6.0 for 72 hours, yielding 79.6 μg/ml.
- Purified rPaDef (95.7% purity) exhibited potent, concentration-dependent antimicrobial activity against Gram-negative and Gram-positive bacteria.
- Bacterial growth was inhibited at concentrations as low as 90 μg/ml.
Conclusions:
- Pichia pastoris serves as an efficient cell factory for expressing and secreting rPaDef.
- This study is the first to report heterologous expression of PaDef in P. pastoris.
- The findings suggest rPaDef holds significant promise for developing novel antibacterial agents.
