Perspectives of inclusion bodies for bio-based products: curse or blessing?

Abstract

The bacterium Escherichia coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body-based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.

The biotechnological production of lacewing silk: From gene to protein‐based material

“…These partially highly pure, intracellularly deposited protein aggregates can, for instance, be purified further using nonionic detergents. [28][29][30][31][32][33][34] Based on prior experiences concerning the processing of silkworm proteins, similar procedures have been developed for recombinant silk proteins, especially spider silk proteins. For example, microspheres, foams, and fibers were generated from artificial spider silk proteins.…”

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications

“…Aiming to improve the production and biological efficiency of this nanomaterial, the increase of the aggregation tendency of proteins of interest is a key aspect and has been evaluated through the use of different aggregation tags, being VP1 [ 6 ], GFIL8 [ 15 ] and ELK16 [ 16 ] three representative examples. Besides, the scale-up of IB production and purification protocols have also been improved during the last decade [ 14 , 17 ]. It has been described that the recombinant protein forming this biomaterial coexists with other proteins such as chaperones, and also with lipids [ 18 , 19 ].…”

Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag

“…Different parameters may impact aggregation such as the extent and kinetics of induced expression, culture conditions (temperature, growth rate, pH) and the presence or absence of folding modulators in the host strain. The same parameters may also affect the size of the inclusion bodies, their physical properties and the balance between denatured and native protein (Rinas et al, 2017;Slouka et al, 2019).…”