Expression and purification of human phosphatase and actin regulator 1 (PHACTR1) in plant-based systems

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Biotechnology

Background:

• Cardiovascular diseases are a major global health concern.
• The protein phosphatase and actin regulator 1 (PHACTR1) is implicated in cardiovascular diseases, but its function is poorly understood.
• PHACTR1 is challenging to express in mammalian cells and aggregates in bacteria, necessitating alternative expression strategies.

Purpose of the Study:

• To evaluate three plant-based expression systems for producing recombinant PHACTR1.
• To optimize conditions for high-yield PHACTR1 expression and recovery.

Main Methods:

• A design of experiments approach was used to test transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves, and BY-2 cell lysate (BYL).
• Optimized extraction buffer conditions, including the use of Triton X-100, were investigated.
• Protein stability and purification methods, including DDK-tag and immobilized metal affinity chromatography, were assessed.

Main Results:

• The BY-2 cell lysate (BYL) system achieved the highest yield of PHACTR1, reaching up to 120 mg/kg biomass equivalent within 48 hours.
• This yield was significantly higher than transient expression in N. benthamiana leaves (1.3-fold increase with p19) and the PCP system (6-fold increase).
• Triton X-100 enhanced PHACTR1 recovery, while biomass blanching and extended storage in raw extracts reduced yield and stability; 15% purity was achieved via IMAC.

Conclusions:

• Plant-based expression, particularly using BY-2 cell lysate, provides a viable and efficient method for producing recombinant PHACTR1.
• Optimized extraction and purification strategies are crucial for maximizing yield and purity of PHACTR1 from plant systems.
• This approach facilitates further research into the role of PHACTR1 in cardiovascular diseases.