Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Human 5-lipoxygenase (5-LO) is crucial for converting arachidonic acid into biologically active leukotrienes.
• Understanding 5-LO function is vital for research into inflammatory and immune responses.

Purpose of the Study:

• To overexpress human 5-lipoxygenase (5-LO) in insect cells using a baculovirus system.
• To characterize the recombinant enzyme's activity and compare it to native 5-LO.
• To investigate the role of specific histidine residues in the enzyme's catalytic activity.

Main Methods:

• Human 5-LO gene cloned into a baculovirus vector for expression in Spodoptera frugiperda insect cells.
• Enzyme activity assays measuring leukotriene synthesis and dependence on Ca2+ and ATP.
• Immunoblot analysis to compare protein levels and size between recombinant and native 5-LO.
• Site-directed mutagenesis of histidine residues (H362S, H372S) and subsequent functional analysis.

Main Results:

• Maximal intracellular 5-LO activity and protein levels detected 48 hours post-infection.
• Recombinant 5-LO synthesized 5-hydroperoxy-icosatetraenoic acid and leukotriene A4 hydrolysis products.
• Enzyme activity showed dependence on Ca2+ and ATP.
• Immunoblots revealed indistinguishable ~80-kDa bands for native and recombinant 5-LO.
• Recombinant expression yielded 50-200 times more 5-LO protein per cell compared to human leukocytes.
• Mutant 5-LO proteins were expressed but showed no substantial alteration in enzyme activity.

Conclusions:

• Baculovirus expression system effectively overexpressed active human 5-lipoxygenase.
• The enzyme requires Ca2+ and ATP for maximal activity.
• Histidine residues 362 and 372 are not essential for the catalytic activity of human 5-lipoxygenase in iron binding.