Stimuli‐responsive peptides and proteins are an exciting class of smart biomaterials for various applications and have received significant attention over the past decades. A wide variety of stimuli such as temperature, pH, ions, enzymes, magnetic field, redox, etc., are explored. This article provides a review of five intensively studied types of stimuli‐responsive peptides and proteins, their design principles and applications, including temperature‐, pH‐, light‐, metal ion‐, and enzyme‐responsive with an emphasis on the key design concepts and switch function. Moreover, typical examples of their applications are discussed to provide a better understanding of the design concept and underlying methodology. This review will facilitate and inspire future innovation toward new peptide‐ and protein‐based materials and their diverse applications.
Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus‐like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein–DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein–DNA complexes dissociate. Capsomeres thus released show enhanced bind‐elute behavior on salt‐tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere–DNA complexes yet enables binding to salt‐tolerant media. Post purification, assembly experiments indicate that DNA–protein interactions can play a negative role during in vitro assembly, as DNA–protein complexes could not be assembled into virus‐like particles, but formed worm‐like structures. This study reveals that the control over DNA–protein interaction is a critical consideration during downstream process development for viral vaccines.
In this study, we present the first integrated and continuous downstream process for the production of microbial virus‐like particle vaccines. Modular murine polyomavirus major capsid VP1 with integrated J8 antigen was used as a model virus‐like particle vaccine. The integrated continuous downstream process starts with crude cell lysate and consists of a flow‐through chromatography step followed by periodic counter‐current chromatography (PCC) (bind‐elute) using salt‐tolerant mixed‐mode resin and subsequent in‐line assembly. The automated process showed a robust behavior over different inlet feed concentrations ranging from 1.0 to 3.2 mg ml−1 with only minimal adjustments needed, and produced continuously high‐quality virus‐like particles, free of nucleic acids, with constant purity over extended periods of time. The average size remained constant between 44.8 ± 2.3 and 47.2 ± 2.9 nm comparable to literature. The process had an overall product recovery of 88.6% and a process productivity up to 2.56 mg h−1 mlresin−1 in the PCC step, depending on the inlet concentration. Integrating a flow through step with a subsequent PCC step allowed streamlined processing, showing a possible continuous pathway for a wide range of products of interest.
The cover image is based on the Article Virus‐like particle preparation is improved by control over capsomere‐DNA interactions during chromatographic purification by Lukas Gerstweiler, Jingxiu Bi, and Anton Peter Jacob Middelberg., https://doi.org/10.1002/bit.27687.
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