efficient-purification-elastin-like-polypeptides-elps-from-e-coli

Efficient Purification of Elastin-Like Polypeptides (ELPs) from <em>E. coli</em> Using an Organic Solvent-based Extraction and Precipitation Method

Elastin-like polypeptides (ELP) are engineered biopolymers built from repetitive pentapeptide sequences that mimic motifs found in mammalian tropoelastin. ...

Purdue University

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore,  studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our  findings. Differential expression of EGFR  was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation.

Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma.

Elastin-like polypeptides (ELP) are a class of materials that are widely used as purification tags and in potential therapeutic applications. We have used the hydrophobic nature of ELP to extract them into organic solvents and precipitate them to obtain highly pure materials. Although many different types of ELP have been rapidly purified in this manner, the underlying mechanism for this process and its ability to retain functional proteins within organic phase-rich media has been unclear. A cleavable ELP-Intein construct fused with the enzyme chorismate mutase (ELP-I-Cm2) was used to better understand the organic solvent extraction process for ELP and the factors impacting the retention of enzyme activity. Our extraction studies indicated that a cell lysis step was essential to stabilize the ELP-I-Cm2 in the organic phase, prevent intein cleavage, and extract the fusion protein with high efficiency and retained activity. Circular dichroism and infrared spectroscopic characterization of ELP-I-Cm2 in organic solvents and aqueous solutions of the extracted and precipitated material indicated that the ELP secondary structure was retained in both environments. Atomic force microscopy and negative stain transmission electron microscopy imaging of ELP-I-Cm2 in organic solvents revealed highly regular circular features that were ∼50 nm in diameter, in contrast to larger (>100 nm) irregular features found in aqueous solutions. Since reverse micelles have often been used in catalytic processes, we evaluated the enzymatic activity of the ELP-I-Cm2 reversed micelles in different organic solvent mixtures and found that Cm2-mediated reactions in organic media were of comparable rate and efficiency to those in aqueous media. Based on these findings, we report an exciting new opportunity for ELP-enzyme fusion applications by exploiting their ability to form catalytically active reverse micelles in organic media.

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of bL LP ucleic acid anoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

High-risk BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) is a condition that is typically treated with Bacillus Calmette-Guérin (BCG) therapy. Unfortunately, NMIBC is characterized by high recurrence, with a significant percentage of BCG patients ultimately requiring radical cystectomy. As a consequence, the development of effective new therapies to avoid RC has become a rapidly evolving field to address this unmet clinical need. To date, three biologics-Keytruda, Adstiladrin, and Anktiva-have been approved by the FDA, and multiple drug modalities, particularly gene therapies, have shown promising results in clinical trials. Advances in drug delivery strategies, such as targeted delivery, sustained release, and permeabilization of protective layers, are critical in overcoming the challenges posed by therapeutic intervention in bladder cancer. This review focuses on high-risk BCG-unresponsive NMIBC therapies that have been or are currently being investigated in clinical trials, offering a broad overview of the delivery system designs and up-to-date clinical outcomes that have been reported as of July 2024. It aims to inform the development of future drug delivery systems for second-line therapies in high-risk BCG-unresponsive NMIBC.

Recent Advances in Drug Delivery Strategies for High-Risk BCG-Unresponsive Non-Muscle Invasive Bladder Cancer: A Brief Review from 2018 to 2024.

Gene delivery has emerged as a groundbreaking technique for altering gene expression, offering new possibilities in treating a vast array of diseases. We report a layer-by-layer elastin-like polypeptide nucleic acid nanoparticle (LENN) system for mRNA delivery as an attractive alternative to viral vectors and lipid nanoparticle (LNP) systems. This study focuses on determining the physical characteristics of LENN bearing mRNA cargo and assessing their biological performance in T24 bladder tumor cells. Our data show that mRNA encoding luciferase forms stable 30 to 130 nm LENN particles via batch mixing to efficiently encapsulate the mRNA strands, are resistant to heparin challenge, and are capable of storage at -20 °C for 3 d as lyophilized powders while retaining full biological activity after rehydration. We also demonstrate that LENN targeted to the epidermal growth factor receptor (EGFR) can efficiently deliver the mRNA cargo to the cytosol of EGFR+ T24 human bladder cancer cells via clathrin-mediated endocytosis where it is translationally active. Lipid profiling analyses show the significant role that upregulated phospholipid biosynthesis plays in nanoparticle internalization and endosomal escape compared to untargeted LENN, indicating the importance of the clathrin pathway in contributing to the delivery efficiency of LENN. Endocytosis inhibition experiments further support the involvement of the clathrin pathway. These findings highlight the compelling features of LENN with respect to their size, in vitro and in vivo targetability, mRNA encapsulation efficiency, complex stability, gene expression, and "green" manufacturability, offering an attractive alternative to existing methods for gene delivery.

Article	Year
Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma. Oncotarget\| PubMed ID: 36082359	2022
Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents. Biomacromolecules\| PubMed ID: 38118170	2024
Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach. Biomacromolecules\| PubMed ID: 39185801	2024
Recent Advances in Drug Delivery Strategies for High-Risk BCG-Unresponsive Non-Muscle Invasive Bladder Cancer: A Brief Review from 2018 to 2024. Pharmaceutics\| PubMed ID: 39339191	2024
Optimizing mRNA delivery with targeted elastin-like polypeptide-based LENN formulations: Insights into the endocytosis mechanism. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 41499406	2026

Saloni Darji

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