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Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze-Thaw Method.

ABSTRACT: Understanding the structure-function of inclusion bodies (IBs) in the last two decades has led to the development of several mild solubilization buffers for the improved recovery of bioactive proteins. The recently developed freeze-thaw-based inclusion body protein solubilization method has received a great deal of attention due to its simplicity and cost-effectiveness. The present report investigates the reproducibility, efficiency, and plausible mechanism of the freeze-thaw-based IB solubilization. The percentage recovery of functionally active protein species of human growth hormone (hGH) and L-asparaginase from their IBs in Escherichia coli and the quality attributes associated with the freeze-thaw-based solubilization method were analyzed in detail. The overall yield of the purified hGH and L-asparaginase protein was found to be around 14 and 25%, respectively. Both purified proteins had functionally active species lower than that observed with commercial proteins. Biophysical and biochemical analyses revealed that the formation of soluble aggregates was a major limitation in the case of tough IB protein like hGH. On the other hand, the destabilization of soft IB protein like L-asparaginase led to the poor recovery of functionally active protein species. Our study provides insight into the advantages, disadvantages, and molecular-structural information associated with the freeze-thaw-based solubilization method.

SUBMITTER: Singhvi P

PROVIDER: S-EPMC8093829 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study investigates the thermal conductivity (λ) and volumetric heat capacity (C) of sandy soil samples under a variety of conditions, including freeze-thaw cycles at temperatures both above and below zero and differing moisture levels. To estimate these thermal properties, a novel predictive model, EFAttNet, was developed, which utilizes custom-designed embedding and attention-based fusion networks. When compared to traditional de Vries empirical models and other baseline algorithms, EFAttNet demonstrated superior accuracy. Preliminary measurements showed that λ values increased linearly with moisture content but decreased with temperature, whereas C values exhibited a rising trend with both moisture content and freezing temperature. Following freeze-thaw cycles, both λ and C were positively influenced by moisture content and freezing temperature. The EFAttNet-based model proved highly accurate in predicting thermal properties, particularly effective at capturing nonlinear relationships among the influencing factors. Among these factors, the degree of saturation had the most significant impact, followed by the number of freeze-thaw cycles, subzero temperatures, porosity, and moisture content. Notably, dry density exerted minimal influence on thermal properties, likely due to the overriding effects of other factors or specific soil characteristics, such as particle size distribution or mineralogical composition. These findings have significant implications for construction and engineering projects, especially in terms of sustainability and energy efficiency. The demonstrated accuracy of the EFAttNet-based model in estimating thermal properties under various conditions holds promise for practical applications. Although focused on specific soil types and conditions, the insights gained can guide further research and development in managing soil thermal properties across diverse environments, thereby enhancing our understanding and application in this field.

Project description:BACKGROUND:The bacterium E. 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. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes. RESULTS:Classical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein. CONCLUSIONS:Time dependent monitoring of IB QA-titer, purity, IB bead size-showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate-time dependently-quality attributes in upstream processing to enable robust downstream processing.

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Molecular Attributes Associated With Refolding of Inclusion Body Proteins Using the Freeze-Thaw Method.

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