rapid-enzymatic-methods-for-amplification-minimal-linear-templates

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins ...

Iowa State University

 For oncolytic virus trials, regulatory agencies often require pharmaceutical industry to evaluate risks of released viruses from patients to environment. This study was to establish a real-time PCR method to assess viral shedding and viral stability in human urine.  Herein, we describe an incubation of viral drug product in human urine and use of real-time PCR as a simple, efficient and high throughput assay to assess the level and stability of a nonenveloped and single stranded RNA virus. The viral stability issue is critical to the collection, transport, storage and testing of clinical samples.  In summary, this simple method provides useful viral stability information at various temperatures and detergents. A similar approach may apply to other RNA viruses (including SARS-CoV-2).

Real-time PCR assay as a simple and efficient tool for viral stability study.

Cell-free protein expression (CFPS) from  cell lysate is an established chemical biology technique. Common efforts to improve synthesis capacity, such as strain engineering and process improvements, have overlooked the opportunity to increase productivity by reducing the dependence on limited, dissolved oxygen. Here we demonstrate conditioning  cells for anaerobic respiration which increases the initial protein expression rate up to 4-fold and increases titer by 50% as compared to traditional aerobic cell lysate when using sfGFP as a reporter protein in CFPS reactions run at atmospheric conditions. This enhancement is even more significant when run in an oxygen-depleted environment, where anaerobic respiration preconditioned cells increase yield when supplemented with nitrite as a terminal electron acceptor (TEA). Furthermore, we test knockout mutants to determine key proteins responsible for enhancing the anaerobically prepared CFPS lysate. Further improvements could be made in preconditioning cells by increasing expression levels of critical pathway enzymes or by screening other TEA.

Jared L. Dopp

Chemical and Biological Engineering

Iowa State University

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Jared L. Dopp

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Chemical and Biological Engineering

Iowa State University

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

Chemical and Biological Engineering