Enhanced soluble protein expression using two new fusion tags.

Area of Science:

• * Molecular Biology
• * Protein Biochemistry
• * Biotechnology

Background:

• * Producing soluble recombinant proteins is crucial for understanding protein function and developing therapeutics.
• * Heterologous protein expression, particularly in Escherichia coli, often results in insoluble protein aggregates.
• * Current methods for improving protein solubility have limitations.

Purpose of the Study:

• * To identify and evaluate novel fusion partners for enhancing the solubility of recalcitrant recombinant proteins.
• * To assess the efficacy of two new fusion tags derived from bacteriophage T7 protein kinase and E. coli Skp.
• * To demonstrate the successful soluble expression and purification of challenging human proteins using these tags.

Main Methods:

• * Construction and expression of fusion proteins using T7 kinase and Skp tags in Escherichia coli.
• * Purification of soluble fusion proteins utilizing an amino-terminal Strep-tag II.
• * Assessment of protein solubility after cleavage of fusion tags using site-specific proteases.

Main Results:

• * Most tested human proteins (Hif1alpha, IL13, folliculin) were expressed in soluble forms when fused with the new tags.
• * Fusion proteins were successfully purified using the integrated Strep-tag II.
• * Some proteins maintained solubility after tag removal, indicating tag-independent solubility enhancement.

Conclusions:

• * The T7 protein kinase and Skp-derived fusion tags are effective tools for improving the soluble expression of difficult proteins.
• * These novel tags offer a promising alternative to existing solubility-enhancing strategies.
• * The developed system facilitates the production of soluble proteins for structural and therapeutic applications.