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Expression of Human Lactoferrin cDNA in Insect Cells
Da-Bing Zhang1, Yu-Lei Jiang, Xiang-Fu Wu
1Shanghai Institute of Plant Physiology, the Chinese Academy of Sciences, Shanghai 200032, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao Acta Biochimica Et Biophysica Sinica
|August 9, 2002
Summary
Researchers developed a recombinant virus to produce human lactoferrin in insect cells. This method successfully secreted functional lactoferrin, showing potential for biotechnological applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Expression
Background:
- Human lactoferrin is a vital iron-binding protein with antimicrobial and immunomodulatory properties.
- Efficient and scalable production of recombinant human lactoferrin is crucial for therapeutic and industrial applications.
Purpose of the Study:
- To construct a recombinant baculovirus for expressing human lactoferrin in insect cells.
- To characterize the expression, secretion, and functional activity of recombinant human lactoferrin.
Main Methods:
- Construction of a transfer plasmid (p8hLFc) with human lactoferrin cDNA.
- Co-transfection into Spodoptera frugiperda (Sf) cells with Autographa californica nuclear polyhedrosis virus (AcNPV BacPAK6) DNA.
- Identification and purification of the recombinant virus (AcNPV-hLFc) using dot hybridization and plaque screening.
- Detection of protein expression via Western blotting and quantification of secreted lactoferrin.
- Purification using affinity chromatography and functional assessment of iron-binding capacity.
Main Results:
- Successful generation and purification of the recombinant AcNPV-hLFc virus.
- Expression of human lactoferrin in Sf cells, constituting approximately 2% of total soluble proteins.
- Secretion of 9.5 mg/L of recombinant lactoferrin into the culture medium.
- Purified recombinant lactoferrin exhibited characteristics similar to human milk lactoferrin on SDS-PAGE.
- Demonstrated iron-binding capacity of the recombinant protein.
Conclusions:
- The baculovirus expression system is effective for producing functional human lactoferrin in insect cells.
- The high yield and demonstrated iron-binding capacity suggest potential for large-scale production.
- This recombinant lactoferrin holds promise for various biotechnological and therapeutic applications.