Purification of Inclusion Bodies Produced in Bacteria and Yeast.

Purification of inclusion bodies (IBs) is gaining importance due to the raising of novel applications for these submicron particulate protein clusters, with potential uses in the biomedical and biotechnological fields among others. Here, we present five optimized methods to purify IBs adapting classical procedures to the material nature, as well as the requirements of the producer cell (Gram-negative bacteria, Gram-positive bacteria, or yeast) and the IB final application.

Bacterial inclusion bodies are industrially exploitable amyloids.

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.

Characterizing the spatio-temporal role of sorting nexin 17 in human papillomavirus trafficking.

The human papillomavirus (HPV) L2 capsid protein plays an essential role during the early stages of viral infection. Previous studies have shown that the interaction between HPV L2 and endosomal sorting nexin 17 (SNX17) is conserved across multiple PV types where it plays an essential role in infectious entry, suggesting an evolutionarily conserved pathway of PV trafficking. Here we show that the peak time of interaction between HPV-16 L2 and SNX17 is rather early, at 2 h post-infection. Interestingly, the L2-SNX17 interaction appears to be important for facilitating capsid disassembly and L1 dissociation, suggesting that L2 recruitment of SNX17 occurs prior to capsid disassembly. Furthermore, we also found evidence of L2-SNX17 association at the later stages of infectious entry, suggesting that the SNX17-mediated sorting machinery is either involved at different stages of HPV trafficking or that L2-SNX17 interaction is a long-lasting event in HPV trafficking.

Bacterial inclusion body purification.

Purification of bacterial inclusion bodies (IBs) is gaining importance due to the raising of novel applications for this type of submicron particulate protein clusters, with potential uses in the biomedical field among others. Here, we present two optimized methods to purify IBs adapting classical procedures to the material nature as well as the requirements of its final application.

Bacterial cell disruption: a crucial step in protein production.

Recombinant protein production significantly improved in the past three decades. Novel expression systems were developed, growth conditions optimised and the technology and thus monitoring and analysis significantly enhanced. However, the studies of bacterial cell disruption were more or less neglected. The existing methods were acceptable until the final product of protein production was soluble and pure protein. However recently, inclusion bodies (IBs) as whole protein particles were also recognised as the final product. Classical methods for bacterial cell disruption are therefore not always suitable, sufficient or even appropriate for isolation of such particulate material. Some of the currently existing methods for bacterial cell disruption were recognised as damaging for the structure of IBs, while sonication was even found harmful for the recombinant protein. The powers needed for disruption of the bacterial cells damage the recombinant proteins and thus their biological activity significantly reduces. Furthermore, the classical isolation methods enable disruption of majority of the bacterial cells and this is enough for isolation of soluble proteins, yet it is not adequate for isolation of particulate material. While remaining bacterial cells sediment together with the IBs, they represent impurity. The need for isolation of cell-free IBs was therefore revealed in the recent studies, because only pure IBs can be used as nanoparticles in further biomedical applications. Therefore it is time to consider, redesign, optimise or even develop new alternative methods that would enable isolation of pure, structurally intact and biologically active particles. Two such alternative methods that enable isolation of bacterial free, active protein particles were developed recently.

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells.

BACKGROUND: We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. RESULTS: K14 IBs were electroporated into SW13 cells grown in culture together with a "reporter" plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. CONCLUSIONS: Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

Active protein aggregates produced in Escherichia coli.

Since recombinant proteins are widely used in industry and in research, the need for their low-cost production is increasing. Escherichia coli is one of the best known and most often used host organisms for economical protein production. However, upon over-expression, protein aggregates called inclusion bodies (IBs) are often formed. Until recently IBs formation represented a bottleneck in protein production as they were considered as deposits of inactive proteins. However, recent studies show that by choosing the appropriate host strain and designing an optimal production process, IBs composed from properly folded and biologically active recombinant proteins can be prepared. Such active protein particles can be further used for the isolation of pure proteins or as whole active protein particles in various biomedical and other applications. Therefore interest in understanding the mechanisms of their formation as well as their properties is increasing.

Reprint of: Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.

Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells.

BACKGROUND: In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry.To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process.To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. RESULTS: In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared.During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation.During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity.High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. CONCLUSIONS: The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

Engineering inclusion bodies for non denaturing extraction of functional proteins.

BACKGROUND: For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-alpha (Truncated form of Tumor necrosis factor alpha with an N-terminal histidine tag) and dN19 LT-alpha (Truncated form of Lymphotoxin alpha). RESULTS: Four structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein.Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7DeltaN6TNF-alpha, and GFP, a significant amount of protein could be extracted from IBs with 0.2% N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied. CONCLUSION: This study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals.

New properties of inclusion bodies with implications for biotechnology.

Human G-CSF (granulocyte colony-stimulating factor) is a well-known biopharmaceutical drug being mostly produced by overexpression in Escherichia coli, where it appears in the form of IBs (inclusion bodies). Following our initial findings that properties of inclusion bodies strongly depend on the growth conditions used, especially growth temperature, we compared the characteristics of the G-CSF inclusion bodies prepared at two different temperatures, namely 42 and 25 degrees C. IBs formed at higher growth temperatures have properties similar to the usually described IBs, containing mainly denatured recombinant protein and being almost completely insoluble in aqueous solutions containing mild detergents or low concentrations of denaturants. In contrast, when produced at lower growth temperature of 25 degrees C, IBs show significantly different properties. Such IBs contain a significant proportion of G-CSF that is easily and directly extractable in the biologically active form, using non-denaturing solutions, which can be exploited for environmentally friendly biotechnological production. Irrespective of the production temperature, a significant decrease in IB volume was observed when transferring IBs from neutral to acidic (around 4) pH. Irreversible contraction of IBs at low pH was documented at the macro- and micro-scopic level using electron microscopy as a characterization tool. Together with volume decrease, a higher density, and thus decreased solubility, of IBs was observed at low pH, resulting in slower and less efficient extractability of the target protein.

Influence of the protein oligomericity on final yield after affinity tag removal in purification of recombinant proteins.

The new aspect concerning the applicability of histidine and other affinity tags for the purification of oligomeric proteins, with particular emphasis on cleavage efficiency and final yield, is presented in this study. The final yield depends on both the cleavage efficiency and the degree of oligomerization of the protein. Cleavage procedures that are good enough for monomeric proteins can be problematic for oligomeric proteins. Random distribution of uncleaved or partially cleaved affinity tags among oligomers is the main cause of reduced yields. A trimeric protein, tumour necrosis factor alpha (TNF-alpha), bearing different histidine tags, was used as a model protein to explore and confirm this theoretical concept. Analysis of mixed TNF trimers, prepared from tag-free TNF doped with various amounts of histidine-tagged TNF, revealed an increased retention of the trimeric protein on immobilized metal-ion affinity chromatography (IMAC) columns. When 20% of histidine-tagged TNF was added, more than 50% of the protein was retained on the IMAC column. Thus, the applicability of histidine- and other affinity tags for purifying oligomeric proteins is significantly prejudiced in the case of higher oligomers. Various histidine-tags were fused to the N-terminus of full-length TNF-alpha and to the truncated form (dN6) of TNF-alpha. Two-step IMAC separation was used for purification. In the first step, IMAC-1, over 95% purity of histidine-tagged protein was achieved in all cases. Endo- and exoproteolytic removal of histidine tags with enterokinase (EKmax) and aminodipeptidase (DAPase) was studied and the major parameters affecting cleavage efficiency, microheterogeneity and final yield are critically discussed. IMAC-2 was used as the second and final step for removing the cleavage enzyme, cleaved tags, unprocessed protein and some other impurities. Selection of the optimal cleavage enzyme depends on the amino acid composition of the N-terminus and the intended use of the purified protein. The main conclusion is that special caution should be taken when introducing affinity tags to oligomeric proteins, with the final goal to produce pure, tag-free protein with acceptable yields. Given the same enzyme cleavage efficiency one can expect progressively reduced final protein yields with increasing degree of oligomerization. This should be considered as a general rule.