-
Current Protein & Peptide Science 2020Heat shock proteins (HSPs) are molecular chaperones involved in a variety of life activities. HSPs function in the refolding of misfolded proteins, thereby contributing... (Review)
Review
Heat shock proteins (HSPs) are molecular chaperones involved in a variety of life activities. HSPs function in the refolding of misfolded proteins, thereby contributing to the maintenance of cellular homeostasis. Heat shock factor (HSF) is activated in response to environmental stresses and binds to heat shock elements (HSEs), promoting HSP translation and thus the production of high levels of HSPs to prevent damage to the organism. Here, we summarize the role of molecular chaperones as anti-heat stress molecules and their involvement in immune responses and the modulation of apoptosis. In addition, we review the potential application of HSPs to cancer therapy, general medicine, and the treatment of heart disease.
Topics: Animals; Antineoplastic Agents; Apoptosis; Benzoquinones; Gene Expression Regulation; Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Male; Myocardial Reperfusion Injury; Oxidative Stress; Plants; Prostatic Neoplasms; Protein Refolding; Response Elements; Signal Transduction; Stress, Physiological
PubMed: 31713482
DOI: 10.2174/1389203720666191111113726 -
Microbial Cell Factories Mar 2015Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from... (Review)
Review
Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
Topics: Escherichia coli; Inclusion Bodies; Models, Molecular; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Recombinant Proteins; Solubility
PubMed: 25889252
DOI: 10.1186/s12934-015-0222-8 -
Nature Mar 2017Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many...
Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.
Topics: Cell Line; Cytosol; HSP70 Heat-Shock Proteins; Heat-Shock Response; Homeostasis; Humans; Mitochondria; Mitochondrial Proteins; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Folding; Protein Refolding; Protein Stability; Protein Transport; Proteins; Proteolysis; Saccharomyces cerevisiae
PubMed: 28241148
DOI: 10.1038/nature21695 -
Journal of Microbiology and... Oct 2016Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide...
Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed into JM109. The transformed cells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.
Topics: Cloning, Molecular; Escherichia coli; Fermentation; Humans; Insulin; Insulin Glargine; Protein Refolding; Recombinant Proteins
PubMed: 27363479
DOI: 10.4014/jmb.1602.02053 -
Biotechnology Advances Dec 2022Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be... (Review)
Review
Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be further purified to obtain pure and active biologicals. This work focusses on refolding as a significant process step during biopharmaceutical manufacturing with an industrial perspective. A theoretical and historical background on protein refolding gives the reader a starting point for further insights into industrial process development. Quality requirements on IBs as starting material for refolding are discussed and further economic and ecological aspects are considered with regards to buffer systems and refolding conditions. A process development roadmap shows the development of a refolding process starting from first exploratory screening rounds to scale-up and implementation in manufacturing plant. Different aspects, with a direct influence on yield, such as the selection of chemicals including pH, ionic strength, additives, etc., and other often neglected aspects, important during scale-up, such as mixing, and gas-fluid interaction, are highlighted with the use of a quality by design (QbD) approach. The benefits of simulation sciences (process simulation and computer fluid dynamics) and process analytical technology (PAT) for seamless process development are emphasized. The work concludes with an outlook on future applications of refolding and highlights open research inquiries.
Topics: Biological Products; Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins
PubMed: 36252795
DOI: 10.1016/j.biotechadv.2022.108050 -
Methods in Molecular Biology (Clifton,... 2023Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as...
Overexpression of heterologous protein in prokaryotic host cells, such as Escherichia coli, usually leads to formation of inactive and insoluble aggregates known as inclusion bodies (IBs). Recovery of refolded and functionally bioactive proteins from IBs is a challenging task, and a unique condition (e.g., solubilizing and refolding buffers) for each individual protein should be experimentally obtained. Here, we present a simple protocol for development of solubilizing and refolding buffers for successful recovery of pure bioactive proteins from IBs.
Topics: Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins; Solubility
PubMed: 36656522
DOI: 10.1007/978-1-0716-2930-7_10 -
Journal of Chromatography. A Apr 2023Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size...
Matrix-assisted refolding (MAR) has been used as an alternative to conventional dilution-based refolding to improve recovery and reduce specific buffer consumption. Size exclusion chromatography (SEC) has been extensively used for MAR because of its ability to load and refold proteins at high concentrations. However, the SEC-based batch MAR processes have the disadvantages of requiring longer columns for better separation and product dilution due to a high column-to-sample volume ratio. In this work, a modified operational scheme is developed for continuous MAR of L-asparaginase inclusion bodies (IBs) using SEC-based periodic counter-current chromatography (PCC). The volumetric productivity of the modified SEC-PCC process is 6.8-fold higher than the batch SEC process. In addition, the specific buffer consumption decreased by 5-fold compared to the batch process. However, the specific activity of the refolded protein (110-130 IU/mg) was less due to the presence of impurities and additives in the refolding buffer. To address this challenge, a 2-stage process was developed for continuous refolding and purification of IBs using different matrices in sequential PCCs. The performance of the 2-stage process is compared with literature reports on single-stage IMAC-PCC and conventional pulse dilution processes for refolding L-asparaginase IBs. The 2-stage process resulted in a refolded protein with enhanced specific activity (175-190 IU/mg) and a high recovery of 84%. The specific buffer consumption (6.2 mL/mg) was lower than the pulse dilution process and comparable to the single-stage IMAC-PCC. A seamless integration of the two stages would considerably increase the throughput without compromising other parameters. High recovery, throughput, and increased operational flexibility make the 2-stage process an attractive option for protein refolding.
Topics: Asparaginase; Chromatography, Gel; Countercurrent Distribution; Protein Folding; Protein Refolding; Proteins; Recombinant Proteins; Inclusion Bodies
PubMed: 37003075
DOI: 10.1016/j.chroma.2023.463938 -
Applied Microbiology and Biotechnology Mar 2021Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In... (Review)
Review
Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In recent years, a change in the mindset regarding IBs could be observed: IBs are no longer considered an unwanted waste product, but a valid alternative to produce a product with high yield, purity, and stability in short process times. However, solubilization of IBs and subsequent refolding is necessary to obtain a correctly folded and active product. This protein refolding process is a crucial downstream unit operation-commonly done as a dilution in batch or fed-batch mode. Drawbacks of the state-of-the-art include the following: the large volume of buffers and capacities of refolding tanks, issues with uniform mixing, challenging analytics at low protein concentrations, reaction kinetics in non-usable aggregates, and generally low re-folding yields. There is no generic platform procedure available and a lack of robust control strategies. The introduction of Quality by Design (QbD) is the method-of-choice to provide a controlled and reproducible refolding environment. However, reliable online monitoring techniques to describe the refolding kinetics in real-time are scarce. In our view, only monitoring and control of re-folding kinetics can ensure a productive, scalable, and versatile platform technology for re-folding processes. For this review, we screened the current literature for a combination of online process analytical technology (PAT) and modeling techniques to ensure a controlled refolding process. Based on our research, we propose an integrated approach based on the idea that all aspects that cannot be monitored directly are estimated via digital twins and used in real-time for process control. KEY POINTS: • Monitoring and a thorough understanding of refolding kinetics are essential for model-based control of refolding processes. • The introduction of Quality by Design combining Process Analytical Technology and modeling ensures a robust platform for inclusion body refolding.
Topics: Inclusion Bodies; Kinetics; Protein Folding; Protein Refolding; Recombinant Proteins; Technology
PubMed: 33598720
DOI: 10.1007/s00253-021-11151-y -
Methods in Molecular Biology (Clifton,... 2022Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major...
Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure-function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.
Topics: Escherichia coli; Inclusion Bodies; Protein Refolding; Proteins; Recombinant Proteins; Solubility
PubMed: 35089569
DOI: 10.1007/978-1-0716-1859-2_22 -
International Journal of Molecular... Jan 2022The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum... (Review)
Review
The prevailing current view of protein folding is the thermodynamic hypothesis, under which the native folded conformation of a protein corresponds to the global minimum of Gibbs free energy . We question this concept and show that the empirical evidence behind the thermodynamic hypothesis of folding is far from strong. Furthermore, physical theory-based approaches to the prediction of protein folds and their folding pathways so far have invariably failed except for some very small proteins, despite decades of intensive theory development and the enormous increase of computer power. The recent spectacular successes in protein structure prediction owe to evolutionary modeling of amino acid sequence substitutions enhanced by deep learning methods, but even these breakthroughs provide no information on the protein folding mechanisms and pathways. We discuss an alternative view of protein folding, under which the native state of most proteins does not occupy the global free energy minimum, but rather, a local minimum on a fluctuating free energy landscape. We further argue that Δ of folding is likely to be positive for the majority of proteins, which therefore fold into their native conformations only through interactions with the energy-dependent molecular machinery of living cells, in particular, the translation system and chaperones. Accordingly, protein folding should be modeled as it occurs in vivo, that is, as a non-equilibrium, active, energy-dependent process.
Topics: Algorithms; Kinetics; Models, Molecular; Models, Theoretical; Protein Conformation; Protein Folding; Protein Refolding; Protein Stability; Proteins; Proteome; Proteomics; Recombinant Proteins; Solubility; Species Specificity; Thermodynamics
PubMed: 35008947
DOI: 10.3390/ijms23010521