Evolved Lactococcus lactis strains for enhanced expression of recombinant membrane proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Production of complex multidomain membrane proteins presents significant challenges for structural genomics.
• A universal method for optimizing membrane protein expression is currently unavailable.

Purpose of the Study:

• To devise a selection method for isolating mutant strains with improved functional expression of recombinant membrane proteins.
• To enhance the yield and folding of membrane proteins for structural and functional studies.

Main Methods:

• A fusion strategy was employed, linking green fluorescent protein and an erythromycin resistance marker (ErmC) to the target protein's C-terminus.
• Selection of improved strains was based on increased erythromycin resistance (enhanced expression) and green fluorescent protein fluorescence (correct folding).
• Directed evolution was used to isolate high-producing mutant strains.

Main Results:

• Three evolved host strains demonstrated 2- to 8-fold increased expression of various proteins.
• Sequencing revealed single-site mutations in the nisK gene (sensor protein of a nisin-A-mediated expression system) in the evolved strains.
• Recombinant membrane protein levels were elevated, with some showing improved folding states.

Conclusions:

• The developed selection method offers a simple, general, and high-throughput approach for directed evolution of protein production.
• This strategy effectively improves yields and folding of recombinant membrane proteins, addressing a critical bottleneck in structural genomics.