Cloning, improved expression and purification of invasion plasmid antigen D (IpaD): an effector protein of enteroinvasive Escherichia coli (EIEC).

The widespread increase in broad-spectrum antimicrobial resistance is making it more difficult to treat gastrointestinal infections. Enteroinvasive Escherichia coli is a prominent etiological agent of bacillary dysentery, invading via the fecal-oral route and exerting virulence on the host via the type III secretion system. IpaD, a surface-exposed protein on the T3SS tip that is conserved among EIEC and Shigella, may serve as a broad immunogen for bacillary dysentery protection. For the first time, we present an effective framework for improving the expression level and yield of IpaD in the soluble fraction for easy recovery, as well as ideal storage conditions, which may aid in the development of new protein therapies for gastrointestinal infections in the future. To achieve this, uncharacterized full length IpaD gene from EIEC was cloned into pHis-TEV vector and induction parameters were optimized for enhanced expression in the soluble fraction. After affinity-chromatography based purification, 61% pure protein with a yield of 0.33 mg per litre of culture was obtained. The purified IpaD was retained its secondary structure with a prominent α-helical structure as well as functional activity during storage, at 4°C, -20°C and -80°C using 5% sucrose as cryoprotectants, which is a critical criterion for protein-based treatments.

Bi-Functional Gold Nanorod-Protein Conjugates with Biomimetic BSA@Folic Acid Corona for Improved Tumor Targeting and Intracellular Delivery of Therapeutic Proteins in Colon Cancer 3D Spheroids.

Gold nanorods (AuNRs) remain well-developed inorganic nanocarriers of small molecules for a plethora of biomedical and therapeutic applications. However, the delivery of therapeutic proteins using AuNRs with high protein loading capacity (LC), serum stability, excellent target specificity, and minimal off-target protein release is not known. Herein, we report two bi-functional AuNR-protein nanoconjugates, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA, supramolecularly coated with folic acid-modified BSA (BSAFA) acting as biomimetic protein corona to demonstrate targeted cytosolic delivery of enhanced green fluorescent protein (EGFP) and therapeutic ribonuclease A enzyme (RNase A) in their functional forms. AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA exhibit high LCs of ∼42 and ∼54%, respectively, increased colloidal stability, and rapid protein release in the presence of biological thiols. As a nanocarrier, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA show resistance to corona formation in high-serum media even after 24 h, guaranteeing a greater circulation lifetime. Folate receptor-targeting BSAFA on the AuNR surface facilitates the receptor-mediated internalization, followed by the release of EGFP and RNase A in HT29 cells. The green fluorescence dispersed throughout the cell's cytoplasm indicates successful cytosolic delivery of EGFP by AuNR@EGFP-BSAFA. AuNR@RNaseA-BSAFA-mediated therapeutic RNase A delivery in multicellular 3D spheroids of HT29 cells exhibits a radical reduction in the cellular RNA fluorescence intensity to 38%, signifying RNA degradation and subsequent cell death. The versatile nanoformulation strategy in terms of the anisotropic particle morphology, protein type, and ability for targeted delivery in the functional form makes the present AuNR-protein nanoconjugates a promising platform for potential application in cancer management.