Core packing defects in an engineered Cro monomer corrected by combinatorial mutagenesis

Area of Science:

• Protein Engineering
• Structural Biology
• Biochemistry

Background:

• The crystal structure of an engineered lambda Cro repressor monomer revealed an expanded hydrophobic core and disordered C-terminal residues.
• This initial structural insight provided a basis for further protein engineering of monomeric Cro variants.

Purpose of the Study:

• To construct and characterize a second generation of monomeric Cro proteins.
• To investigate the effects of mutations in the hydrophobic core and C-terminal residues on protein stability and expression.

Main Methods:

• Combinatorial mutagenesis of selected core and C-terminal residues in the cro gene.
• Genetic screening in Escherichia coli for clones with high protein accumulation.
• Gene sequencing, protein purification, and analysis of thermal stability and expression levels.

Main Results:

• Alternative arrangements of hydrophobic core residues led to significant increases in thermal stability.
• Mutations at the C-terminus had minimal impact on protein stability but enhanced expression levels.
• Successfully identified engineered Cro variants with improved properties.

Conclusions:

• Hydrophobic core residues are critical for enhancing the thermal stability of monomeric Cro proteins.
• C-terminal modifications can be utilized to optimize protein expression levels.
• This study demonstrates a successful strategy for protein engineering of lambda Cro repressor for improved stability and expression.