Biotechnological applications of recombinant single-domain antibody fragments

<p>Abstract</p> <p>Background</p> <p>Single-domain antibody fragments possess structural features, such as a small dimension, an elevated stability, and the singularity of recognizing epitopes non-accessible for conventional antibodies that make them interesting for several research and biotechnological applications.</p> <p>Results</p> <p>The discovery of the single-domain antibody's potentials has stimulated their use in an increasing variety of fields. The rapid accumulation of articles describing new applications and further developments of established approaches has made it, therefore, necessary to update the previous reviews with a new and more complete summary of the topic.</p> <p>Conclusions</p> <p>Beside the necessary task of updating, this work analyses in detail some applicative aspects of the single-domain antibodies that have been overseen in the past, such as their efficacy in affinity chromatography, as co-crystallization chaperones, protein aggregation controllers, enzyme activity tuners, and the specificities of the unconventional single-domain fragments.</p

A step ahead: combining protein purification and correct folding selection

The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression

Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli

Bacteria are simple and cost effective hosts for producing recombinant proteins. However, their physiological features may limit their use for obtaining in native form proteins of some specific structural classes, such as for instance polypeptides that undergo extensive post-translational modifications. To some extent, also the production of proteins that depending on disulfide bridges for their stability has been considered difficult in E. coli

Minimal information: an urgent need to assess the functional reliability of recombinant proteins used in biological experiments

Structural characterization of proteins used in biological experiments is largely neglected. In most publications, the information available is totally insufficient to judge the functionality of the proteins used and, therefore, the significance of identified protein-protein interactions (was the interaction specific or due to unspecific binding of misfolded protein regions?) or reliability of kinetic and thermodynamic data (how much protein was in its native form?). As a consequence, the results of single experiments might not only become questionable, but the whole reliability of systems biology, built on these fundaments, would be weakened

Characterization of the aggregates formed during recombinant protein expression in bacteria

BACKGROUND: The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates. RESULTS: The total lysate separated into 4 different fractions whereas only the one with the lowest density was detected when the supernatant recovered after ultracentrifugation was loaded onto the sucrose gradient. The three further aggregate sub-classes were otherwise indistinctly precipitated in the pellet. The distribution of the recombinant protein among the four subclasses was strongly dependent on the DnaK availability, with larger aggregates formed in Dnak(- )mutants. The aggregation state of the GFP-GST recovered from each of the four fractions was further characterized by examining three independent biochemical parameters. All of them showed an increased complexity of the recombinant protein aggregates starting from the top of the sucrose gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also confirmed by electron microscopy analysis of the macro-structure formed by the different aggregates. Large fibrils were rapidly assembled when the recombinant protein was incubated in the presence of cellular extracts, but the GFP-GST fusion purified soon after lysis failed to undergo amyloidation, indicating that other cell components probably participate in the active formation of large aggregates. Finally, we showed that aggregates of lower complexity are more efficiently disaggregated by a combination of molecular chaperones. CONCLUSION: An additional analytical tool is now available to investigate the aggregation process and separate subclasses by their mass. It was possible to demonstrate the complexity of the aggregation pattern of a recombinant protein expressed in bacteria and to characterize biochemically the different aggregate subclasses. Furthermore, we have obtained evidence that the cellular environment plays a role in the development of the aggregates and the problem of the artifact generation of aggregates has been discussed using in vitro models. Finally, the possibility of separating aggregate fractions with different complexities offers new options for biotechnological strategies aimed at improving the yield of folded and active recombinant proteins

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors

BACKGROUND: The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC). RESULTS: A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods. CONCLUSION: The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression

Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and VHH domains

BACKGROUND: Recombinant antibodies from Camelidae (VHHs) are potentially useful tools for both basic research and biotechnological applications because of their small size, robustness, easy handling and possibility to refold after chemio-physical denaturation. Their heat tolerance is a particularly interesting feature because it has been recently related to both high yields during recombinant expression and selective purification of folded protein. RESULTS: Purification of recombinant RE3 VHH by heat treatment yielded the same amount of antibody as purification by affinity chromatography and negligible differences were found in stability, secondary structure and functionality. Similar results were obtained using another class of thermotolerant proteins, the single domain VH scaffold, described by Jespers et al. [8]. However, thermosensitive VHs could not withstand the heat treatment and co-precipitated with the bacterial proteins. In both cases, the thermotolerant proteins unfolded during the treatment but promptly refolded when moved back to a compatible temperature. CONCLUSION: Heat treatment can simplify the purification protocol of thermotolerant proteins as well as remove any soluble aggregate. Since the re-folding capability after heat-induced denaturation was previously correlated to higher performance during recombinant expression, a unique heating step can be envisaged to screen constructs that can provide high yields of correctly-folded proteins

Supplementary material for the article: Popovic, M.; Mazzega, E.; Toffoletto, B.; de Marco, A. Isolation of Anti-Extra-Cellular Vesicle Single-Domain Antibodies by Direct Panning on Vesicle-Enriched Fractions. Microbial cell factories 2018, 17 (1), 6. https://doi.org/10.1186/s12934-017-0856-9

Supplementary material for: [https://doi.org/10.1186/s12934-017-0856-9]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2069

Monodispersity of recombinant Cre recombinase correlates with its effectiveness in vivo

<p>Abstract</p> <p>Background</p> <p>Cre recombinase is a common reagent used for the <it>in vivo </it>on/off switching of the expression of target genes flanked by <it>loxP </it>sites. In particular, recombinant TAT-Cre fusion constructs purified from bacteria have been used to promote the cell uptake of the enzyme. However, the recovery of active TAT-Cre remains a demanding process and its specific activity varies significantly among batches, making difficult data comparison.</p> <p>Results</p> <p>We noticed a strong correlation between recombinase activity and enzyme monodispersity. The existence of such correlation enabled us to indirectly monitor the TAT-Cre recombinase activity during the multi-step purification process by measuring its monodispersity, a parameter detectable by means of a spectrofluorimetric assay that allows the calculation of the Aggregation Index (AI) in an easy and rapid way. AI values were recorded after each purification passage to identify the critical steps and to choose optimal alternatives for chromatographic conditions, desalting procedures, and protocols for bacterial endotoxin removal. Furthermore, the effect of metal ions and temperature on TAT-Cre aggregation and inactivation was characterized <it>in vitro</it>. Finally, we optimized the enzyme delivery protocol <it>in vivo </it>by following the accumulation tuning of the reporter protein β-catenin.</p> <p>Conclusion</p> <p>A rational purification protocol for TAT-Cre has been developed by choosing the options that minimize the enzyme aggregation. Our data suggest that AI measurement should support the optimization of any protocol aiming at the recovery of monodispersed protein.</p

Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli

<p>Abstract</p> <p>Background</p> <p>The overproduction of recombinant proteins in host cells often leads to their misfolding and aggregation. Previous attempts to increase the solubility of recombinant proteins by co-overproduction of individual chaperones were only partially successful. We now assessed the effects of combined overproduction of the functionally cooperating chaperone network of the <it>E. coli </it>cytosol on the solubility of recombinant proteins.</p> <p>Results</p> <p>A two-step procedure was found to show the strongest enhancement of solubility. In a first step, the four chaperone systems GroEL/GroES, DnaK/DnaJ/GrpE, ClpB and the small HSPs IbpA/IbpB, were coordinately co-overproduced with recombinant proteins to optimize <it>de novo </it>folding. In a second step, protein biosynthesis was inhibited to permit chaperone mediated refolding of misfolded and aggregated proteins <it>in vivo</it>. This novel strategy increased the solubility of 70% of 64 different heterologous proteins tested up to 42-fold.</p> <p>Conclusion</p> <p>The engineered <it>E. coli </it>strains and the two-step procedure presented here led to a remarkable increase in the solubility of a various recombinant proteins and should be applicable to a wide range of target proteins produced in biotechnology.</p