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PCR cloning and baculovirus expression of human lactoperoxidase and myeloperoxidase
K Shin1, H Hayasawa, B Lönnerdal
1Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd., 5-1-83 Higashihara, Zama, Kanagawa, 228-8583, Japan.
Abstract:
Lactoperoxidase (LPO) and myeloperoxidase (MPO) have been identified previously in human milk. These peroxidases have antimicrobial activity and presumably contribute to the protective functions of milk. In this study, we amplified genes encoding LPO and MPO from human mammary gland cDNA by the polymerase chain reaction (PCR). These genes were expressed in a baculovirus-insect cell system. Peroxidase activity was observed in the culture supernatant of Tricoplusia ni cells infected with the recombinant viruses and the levels increased upon addition of delta-aminolevulinic acid. Purified recombinant human LPO and MPO, both with a molecular mass of about 80 kDa, showed properties similar to bovine LPO and human MPO, respectively, in terms of absorption spectrum, sensitivity to dapsone, specificity for chloride ions, and reactivity with anti-bovine LPO or anti-MPO antibodies. Our data suggest that this expression system is useful for studying the catalytic mechanism and biological significance of these human peroxidases.
Insights
Researchers expressed human lactoperoxidase (LPO) and myeloperoxidase (MPO) genes in insect cells. This system successfully produced active peroxidases, aiding the study of their antimicrobial roles in human milk.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Human milk contains lactoperoxidase (LPO) and myeloperoxidase (MPO), enzymes with known antimicrobial functions.
- These peroxidases are believed to contribute to the protective properties of human milk for infants.
Purpose of the Study:
- To amplify and express genes encoding human LPO and MPO using a recombinant baculovirus-insect cell system.
- To characterize the functional properties of the recombinant human peroxidases.
- To evaluate the utility of this expression system for studying human LPO and MPO.
Main Methods:
- Polymerase chain reaction (PCR) amplification of LPO and MPO genes from human mammary gland cDNA.
- Expression of recombinant human LPO and MPO in Tricoplusia ni insect cells using a baculovirus system.
- Purification and characterization of recombinant peroxidases, including activity assays and spectral analysis.
Main Results:
- Functional recombinant human LPO and MPO were successfully expressed and secreted into the culture supernatant.
- Peroxidase activity was enhanced by the addition of delta-aminolevulinic acid.
- Purified recombinant enzymes (approx. 80 kDa) exhibited properties analogous to native bovine LPO and human MPO.
Conclusions:
- The baculovirus-insect cell expression system is effective for producing active human LPO and MPO.
- This system provides a valuable tool for investigating the catalytic mechanisms and biological significance of these important human milk peroxidases.