Protein aggregated into bacterial inclusion bodies does not result in protection from proteolytic digestion

Proteolytic resistance, as conferred by protein aggregation into inclusion bodies, has not been explored in detail. We have investigated the eventual digestion of several closely-related proteins, namely six insertional and two fusion mutants of the homotrimeric bacteriophage P22 tailspike (TSP) protein. When over-produced in E. coli, all these polypeptides form inclusion bodies accompanied by only traces of soluble protein. The mutations introduced in TSP impaired its degradation and enhanced its half live up to ten-fold, without affecting protein solubility. This indicates that protein properties other than solubility, are the main determinants of susceptibility to proteolysis. In addition, the analysis of the degradation fragments strongly suggests that the aggregated TSP polypeptides undergo a site-limited proteolytic attack, and that their complete digestion occurs through an in situ cascade cleavage process.     

Inverse sequence similarity of proteins does not imply structural similarity

There is a debate on the folding of proteins with inverted sequences. Theoretical approaches and experiments give contradictory results. Many proteins in the Protein Data Bank (PDB) show conspicuous inverse sequence similarity (ISS) to each other. Here we analyze whether this ISS is related to structural similarity. For the first time, we performed a large scale three-dimensional (3-D) superposition of corresponding Calpha atoms of forwardly and inversely aligned proteins and tested the degree of secondary structure identity between them. Comparing proteins of less than 50% pairwise sequence identity, only 0.5% of the inversely aligned pairs had similar folds (99 out of 19073), whereas about 9% of forwardly aligned proteins in the same score and length range show similar 3-D structures (1731 out of 19248). This observation strongly supports the view that the inversion of sequences in almost all cases leads to a different folding property of the protein. Inverted sequences are thus suitable as protein-like sequences for control purposes without relations to existing proteins.     

Protein aggregation as bacterial inclusion bodies is reversible

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.     

Renaturation of recombinant proteins produced as inclusion bodies

Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies. Within the last few years specific methods and strategies have been developed to prepare active proteins from these inclusion bodies. These methods include (i) isolation of inclusion bodies after disintegration of cells by mechanical forces and purification by washing with detergent solutions or low concentrations of denaturant, (ii) solubilization of inclusion bodies with high concentrations of urea or guanidine-hydrochloride in combination with reducing reagents, and (iii) renaturation of the proteins including formation of native disulphide bonds. Renatured and native disulphide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulphide interchange starting from mixed disulphides containing peptides. The final yield of renatured proteins can be increased by adding low concentrations of denaturant during renaturation.     

Construction and deconstruction of bacterial inclusion bodies

Bacterial inclusion bodies (IBs) are refractile aggregates of protease-resistant misfolded protein that often occur in recombinant bacteria upon gratuitous overexpression of cloned genes. In biotechnology, the formation of IBs represents a main obstacle for protein production since even favouring high protein yields, the in vitro recovery of functional protein from insoluble deposits depends on technically diverse and often complex re-folding procedures. On the other hand, IBs represent an exciting model to approach the in vivo analysis of protein folding and to explore aggregation dynamics. Recent findings on the molecular organisation of embodied polypeptides and on the kinetics of inclusion body formation have revealed an unexpected dynamism of these protein aggregates, from which polypeptides are steadily released in living cells to be further refolded or degraded. The close connection between in vivo protein folding, aggregation, solubilisation and proteolytic digestion offers an integrated view of the bacterial protein quality control system of which IBs might be an important component especially in recombinant bacteria.     

Amyloid-like properties of bacterial inclusion bodies

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.     

Isolation of cell-free bacterial inclusion bodies

Background  

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.     

Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

ABSTRACT: A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.     

Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies

Overexpression of cloned or synthetic genes in Escherichia coli often results in the formation of insoluble protein inclusion bodies. Within the last decade, specific methods and strategies have been developed for preparing active recombinant proteins from these inclusion bodies. Usually, the inclusion bodies can be separated easily from other cell components by centrifugation, solubilized by denaturants such as guanidine hydrochloride (Gdn-HCl) or urea, and then renatured through a refolding process such as dilution or dialysis. Recent improvements in renaturation procedures have included the inhibition of aggregation during refolding by application of low molecular weight additives and matrix-bound renaturation. These methods have made it possible to obtain high yields of biologically active proteins by taking into account process parameters such as protein concentration, redox conditions, temperature, pH, and ionic strength.     

Towards revealing the structure of bacterial inclusion bodies

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.     

Localization of functional polypeptides in bacterial inclusion bodies

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a beta-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular beta-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.     

Towards revealing the structure of bacterial inclusion bodies

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6–12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.Key words: bacterial, inclusion bodies, amyloid fibrils, protein aggregation, amyloid-like, nuclear magnetic resonance, electron microscope, X-ray diffraction, hydrogen/deuterium exchange, cross-β     

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between "yield" and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially "screen," in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts.     

Localization of chaperones DnaK and GroEL in bacterial inclusion bodies

By immunostaining and transmission electron microscopy, chaperones DnaK and GroEL have been identified at the solvent-exposed surface of bacterial inclusion bodies and entrapped within these aggregates, respectively. Functional implications of this distinct localization are discussed in the context of Escherichia coli protein quality control.     

Surface inactivation of bacterial viruses and of proteins

1. The seven bacterial viruses of the T group active against E. coli, are rapidly inactivated at gas-liquid interfaces. 2. The kinetics of this inactivation whether brought about by shaking or by bubbling with nitrogen are those of a first order reaction. 3. This inactivation may be prevented by the addition of enough protein to maintain the gas-liquid interface in a saturated condition. 4. The analogy between this phenomenon and the surface denaturation of proteins is pointed out and discussed.     

Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study

Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end‐to‐end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three‐column periodic counter‐current chromatography for capture of the product followed by a three‐column con‐current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end‐to‐end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998–1009, 2017     

In situ protein folding and activation in bacterial inclusion bodies

Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.