Engineering inclusion bodies for non denaturing extraction of functional proteins

<p>Abstract</p> <p>Background</p> <p>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-α (Truncated form of Tumor necrosis factor α with an N-terminal histidine tag) and dN19 LT-α (Truncated form of Lymphotoxin α).</p> <p>Results</p> <p>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.</p> <p>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, His7ΔN6TNF-α, 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.</p> <p>Conclusion</p> <p>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.</p

In vivo and in vitro Cleavage of Glucoamylase-TNFα Fusion Protein Secreted from Aspergillus niger

The most common expression strategy for secreting heterologous proteins from filamentous fungus Aspergillus niger is based on fusion with glucoamylase gene which contains cleavage site for kexin protease (KEX2). However, secretion of recombinant proteins in the form of a fusion-protein without a host-specific cleavage site is usually higher than secretion of the mature protein obtained after in vivo cleavage. We tried to take advantage of such a higher production by cleaving the fusion protein in vitro after fermentation, instead of in vivo during secretion. Similar level of production as after in vivo cleavage was found when human tumor necrosis factor α (TNFα) was produced as a fusion protein with glucoamylase having the enterokinase cleavage site. In addition to the correctly processed TNFα, some non-specific cleavage was observed, which resulted in a shortened N-terminus. This was still better than in vivo cleavage where only truncated forms of TNFα were obtained. Although the fusion protein was cleaved by enterokinase directly in the medium before purification, this shorter N-terminus was probably a consequence of aberrant enterokinase cleavage. Isolation of fusion protein with His-tag by affinity chromatography with immobilized metal chelate (although normally fast and easy) was not possible because the sequence of five consecutive histidines attached to the N-terminus of the glucoamylase fusion partner was completely cleaved off by proteolysis

Attachment of Histidine Tags to Recombinant Tumor Necrosis Factor-Alpha Drastically Changes Its Properties

When studying two different histidine tags attached to the N-termini of the trimeric cytokine tumor necrosis factor alpha (TNF), the biological activity — measured as cytotoxicity on the L-929 cell line — of both tagged proteins was drastically reduced. The longer His10 tag reduced cytotoxicity to approximately 16% and the shorter His7 tag to 6% of the activity of their nontagged counterparts. After removal of the tags, biological activities reverted to the expected normal values, which clearly shows the key role of the attached histidine tags in diminishing biological activity. Studies on the mechanism of these effects revealed no specific interactions and showed that even the natural flexible N-terminus of TNF presents a steric hindrance for receptor binding, while any extension of the N-terminus increases this hindrance and consequently reduces biological activity. Also, in other proteins, the ligand or substrate binding sites may be hindered by histidine tags, leading to wrong conclusions about biological activity or other properties of the proteins. Thus caution is advised when using His-tagged proteins directly in screening procedures or in research

Cellulose: from biocompatible to bioactive material

International audienceSince the papyri, cellulose has played a significant role in human culture, especially as paper. Nowadays, this ancient product has found new scientific applications in the expanding sector of paper-based technology. Among paper-based devices, paper-based biosensors raise a special interest. The high selectivity of biomolecules for target analytes makes these sensors efficient. Moreover, simple paper-based detection devices do not require hardware or specific technical skill. They are inexpensive, rapid, user-friendly and therefore highly promising for providing resource-limited settings with point-of-care diagnostics. The immobilization of biomolecules onto cellulose is a key step in the development of these sensing devices. Following an overview of cellulose structural features and physicochemical properties, this article reviews current techniques for the immobilization of biomolecules on paper membranes. These procedures are categorized into physical, biological and chemical approaches. There is no universal method for biomolecule immobilization. Thus, for a given paper-based biochip, each strategy can be considered

Importance of the Support Properties for Immobilization or Purification of Enzymes

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R, Colciencia (Colombia) and CNPq (Brazil). The predoctoral fellowships for Mr dos Santos (CNPq, Brazil) are also recognized. Á. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovaciûn for a Ramón y Cajal fellowship (RyC-2009-03813)

G-CSF/anti-G-CSF antibody complexes drive the potent recovery and expansion of CD11b+Gr-1+ myeloid cells without compromising CD8+ T cell immune responses

BACKGROUND: Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. METHODS: We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. RESULTS: We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b(+)Gr-1(+) myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8(+) T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. CONCLUSIONS: Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF