An in vivo approach to isolating allosteric pathways using hybrid multimeric proteins

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Allosteric interactions in native tetramers of Escherichia coli phosphofructokinase (EcPFK) are complex.
• Previous in vitro methods for generating EcPFK hybrids involved time-consuming dissociation and re-association steps, resulting in low yields.

Purpose of the Study:

• To develop a more efficient in vivo method for producing hybrid tetramers of EcPFK.
• To increase hybrid yields and decrease isolation time compared to existing in vitro techniques.

Main Methods:

• Co-expression of wild-type and mutant EcPFK genes in E. coli using distinct plasmids (pALTER-Ex2 and pALTER-1).
• Site-directed mutagenesis to create mutant EcPFK genes.
• Affinity purification and anion-exchange chromatography to separate hybrid species (4:0, 3:1, 2:2, 1:3, 0:4).
• Optimization of expression vector and charge-tag mutations to improve yields of specific hybrids.

Main Results:

• Successfully co-expressed and purified hybrid tetramers of EcPFK in vivo.
• Identified all five hybrid species, though 1:3 and 0:4 hybrids were initially in low amounts.
• Substantially increased the yield of the 1:3 hybrid by modifying the expression vector and mutations.
• Demonstrated that the in vivo method is faster and yields more hybrids than the in vitro approach.

Conclusions:

• The in vivo co-expression method provides a significantly improved approach for generating EcPFK hybrid tetramers.
• This optimized method enhances hybrid yields and reduces the time required for isolation, facilitating further studies of EcPFK allosteric regulation.