Optimized bacterial expression of human apolipoprotein A-I.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Expression

Background:

• Apolipoprotein A-I (apoA-I) is crucial for high-density lipoprotein structure and reverse cholesterol transport.
• Understanding apoA-I structure-function relationships requires efficient recombinant protein production.
• Existing methods for producing recombinant apoA-I have limitations in yield and purification.

Purpose of the Study:

• To optimize a bacterial expression system for high-yield production of recombinant wild-type and mutant human apolipoprotein A-I (apoA-I).
• To facilitate structure-function studies by improving the ease of purification and enabling site-specific modifications.
• To develop a modified apoA-I cDNA and expression protocol for enhanced protein yields.

Main Methods:

• Introduction of silent mutations into the apoA-I cDNA to optimize codon usage in Escherichia coli.
• Modification of the apoA-I coding sequence to eliminate codons with low tRNA abundance.
• Site-directed mutagenesis to introduce an acid-labile bond for His-Tag cleavage and purification.

Main Results:

• Engineered apoA-I cDNA yielded 100+/-20 mg/L of recombinant protein, a fivefold increase over previous methods.
• Silent mutations improved expression by optimizing codon usage and eliminating low-abundance tRNAs.
• A Glu2Asp mutation enabled efficient N-terminal His-Tag removal via acid cleavage, yielding pure apoA-I.

Conclusions:

• The optimized bacterial expression system significantly enhances recombinant apoA-I yield.
• The modified cDNA and purification strategy facilitate structure-function studies of apoA-I.
• This improved method provides a robust platform for producing wild-type and mutant apoA-I.