Expression and Purification of the Cell-Penetrating Peptide MAP Fused to Protein Cargo

Abstract

Candida albicans is a common fungal pathogen that presents a significant public health issue due to its resistance to traditional antifungal small molecule medications. While large therapeutics offer potential as alternative treatments with novel mechanisms of action, their efficacy is hindered by low cellular uptake and limited accessibility to intracellular targets. Cell-penetrating peptides (CPPs) are promising vehicles for facilitating the intracellular delivery of large biomolecules. CPPs are short protein peptides capable of crossing various membrane barriers, even when conjugated to cargo molecules. However, producing CPP fusion proteins can be challenging due to their higher toxicity to host cells. To enhance expression, we studied and optimized several contributing factors, including expression partners, host cell strains, sequential order of CPP and cargo, and induction conditions, for the fusion of CPP MAP to the cargo monomeric enhanced green fluorescent protein (meGFP). Our findings indicated that the expression partner, combined with positioning MAP at the N-terminus of the cargo, resulted in relatively high expression levels. The highest expression level was achieved in the BL21(DE3) Escherichia coli strain, with induction at 20°C for 24 hours. These results support further research on the application of MAP recombinant proteins and lay the foundation for the generalization of CPP recombinant protein expression.