[Optimized inverse PCR strategy for constructing multilocus mutants efficiently].

Area of Science:

• Molecular Biology
• Protein Engineering
• Biotechnology

Background:

• Protein mutants are crucial for understanding protein structure and function.
• Existing methods for constructing multi-site mutants can be inefficient and time-consuming.

Purpose of the Study:

• To develop an efficient and rapid method for constructing multi-site protein mutants.
• To simplify the process of generating mutations in adjacent amino acid residues.

Main Methods:

• Design of long and short primers with specific mutation site and annealing temperature parameters.
• Two sets of reverse PCR amplifications to generate non-methylated linear plasmids.
• DpnⅠ digestion, denaturation-annealing of linear plasmids, and transformation into Escherichia coli competent cells.

Main Results:

• Successfully mutated 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously.
• The method simplifies the construction of multi-site mutants.
• Increased efficiency in protein structure and function research.

Conclusions:

• The developed method provides an efficient and rapid approach for multi-site mutant construction.
• This technique significantly enhances the study of protein structure and function by simplifying mutant generation.