Expression of normal and mutagenized apolipoprotein CII in procaryotic cells. Structure-function relationship

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Apolipoprotein C-II (apoCII) is essential for activating lipoprotein lipase, a key enzyme in lipid metabolism.
• Previous studies lacked a complete understanding of the apoCII structure-function relationship, particularly the domain responsible for lipase activation.

Purpose of the Study:

• To construct a full-length human apoCII cDNA clone.
• To express and characterize mature apoCII and its functional domains.
• To identify the specific apoCII domain responsible for activating serum lipoprotein lipase.

Main Methods:

• Construction of a full-length human apoCII cDNA using synthetic oligonucleotides.
• In vitro transcription and translation of the apoCII cDNA.
• Expression of apoCII as a fusion protein (apoCII-beta-galactosidase) in E. coli.
• Generation and expression of C-terminal deletion mutants using Bal31 nuclease digestion.

Main Results:

• A complete human apoCII cDNA clone was successfully constructed and expressed.
• The N-terminal signal sequence of apoCII was accurately cleaved during in vitro translation.
• Expression in E. coli yielded processed apoCII, and deletion mutants allowed for the identification of the lipase-activating domain.

Conclusions:

• The study successfully produced a full-length human apoCII clone and expressed functional protein.
• The C-terminal domain of apoCII was identified as responsible for the activation of serum lipoprotein lipase.
• This work provides a foundation for further structural and functional studies of apoCII.