Functional recombinant apolipoprotein A5 that is stable at high concentrations at physiological pH

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Apolipoprotein A5 (APOA5) is vital for human triglyceride metabolism.
• APOA5 dysfunction is linked to hypertriglyceridemia, obesity, and cardiovascular diseases.
• Previous bacterial production of APOA5 yielded low amounts and poor solubility, hindering research.

Purpose of the Study:

• To enhance the yield and solubility of recombinant Apolipoprotein A5 (APOA5).
• To establish a robust system for producing soluble APOA5 for structure-function studies.
• To investigate the physical properties and functionality of the produced APOA5.

Main Methods:

• Engineered a fusion protein construct using a codon-optimized expression vector.
• Optimized an efficient protein refolding protocol.
• Screened buffer systems at physiological pH to maximize solubility and yield.

Main Results:

• Achieved a high yield (25 mg/l) of lipid-free APOA5 using a bacterial expression system.
• Produced APOA5 soluble at concentrations up to 10 mg/ml in bicarbonate buffers at pH 7.8.
• Characterized APOA5 as multimers in solution, with pH-dependent changes in alpha-helicity.
• Demonstrated APOA5 functionality in binding and emulsifying lipid vesicles and inhibiting TG accumulation in vivo.

Conclusions:

• Developed a high-yield, cost-effective bacterial expression system for soluble APOA5.
• The produced APOA5 is suitable for biochemical and structural studies.
• This system facilitates further research into APOA5's role in lipid metabolism and related diseases.