Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

<p>Abstract</p> <p>Background</p> <p>Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library.</p> <p>Results</p> <p>Both phages and <it>E. coli </it>cells pass non-hindered through the interconnected pores of macroporous gel, so called <it>cryogel</it>. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. <it>E. coli </it>cells were applied on the column for infection with the specifically bound phages.</p> <p>Conclusion</p> <p>Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection.</p

Evaluating predictive markers for viral rebound and safety assessment in blood and lumbar fluid during HIV-1 treatment interruption

Background: Validated biomarkers to evaluate HIV-1 cure strategies are currently lacking, therefore requiring analytical treatment interruption (ATI) in study participants. Little is known about the safety of ATI and its long-term impact on patient health. Objectives: ATI safety was assessed and potential biomarkers predicting viral rebound were evaluated. Methods: PBMCs, plasma and CSF were collected from 11 HIV-1-positive individuals at four different timepoints during ATI (NCT02641756). Total and integrated HIV-1 DNA, cell-associated (CA) HIV-1 RNA transcripts and restriction factor (RF) expression were measured by PCR-based assays. Markers of neuroinflammation and neuronal injury [neurofilament light chain (NFL) and YKL-40 protein] were measured in CSF. Additionally, neopterin, tryptophan and kynurenine were measured, both in plasma and CSF, as markers of immune activation. Results: Total HIV-1 DNA, integrated HIV-1 DNA and CA viral RNA transcripts did not differ pre- and post-ATI. Similarly, no significant NFL or YKL-40 increases in CSF were observed between baseline and viral rebound. Furthermore, markers of immune activation did not increase during ATI. Interestingly, the RFs SLFN11 and APOBEC3G increased after ATI before viral rebound. Similarly, Tat-Rev transcripts were increased preceding viral rebound after interruption. Conclusions: ATI did not increase viral reservoir size and it did not reveal signs of increased neuronal injury or inflammation, suggesting that these well-monitored ATIs are safe. Elevation of Tat-Rev transcription and induced expression of the RFs SLFN11 and APOBEC3G after ATI, prior to viral rebound, indicates that these factors could be used as potential biomarkers predicting viral rebound

The elusive source of HIV-1 rebound after treatment interruption

Identifying the source of viral rebound during a monitored analytical treatment interruption (ATI) would reveal potential targets for cure strategies. Therefore, we examined the genetic composition of proviral DNA in different subsets from participants on antiretroviral therapy and compared this to rebounding virus after an ATI. Eleven participants underwent a monitored ATI and were sampled from different anatomical sites prior to and after the ATI. From the peripheral blood, Naïve (TNA), central (TCM), transitional (TTM) and effector (TEM) memory CD4+ T cells were sorted as were CD45 cells from gut-associated lymphoid tissue (GALT). Using single-genome sequencing (SGS) the env region of HIV DNA and plasma-derived RNA was sequenced. In an ongoing study, Full-Length Individual Proviral Sequencing (FLIPS) and Integration Site Loop Amplification (ISLA) assays were performed on the T cell subsets from 2 participants. For participant STAR10, 87 integration sites (IS) and 113 proviral genomes were sequenced while only 3 unique intact proviruses (3%) were identified. A cluster of 17 identical defective proviruses were linked to an IS (9% of all IS) in STAT5B located in TCM, TNA, TEM and TTM. When comparing the FLIPS to SGS env sequences a 100% match was found between one defective provirus and one plasma HIV RNA sequence after rebound. For participant STAR11, 37 IS and 105 proviral genomes were sequenced yielding 14 intact proviruses (13%) with the highest proportion found predominantly in the TEM subset (n=13, 45%). Four different clusters of identical sequences could be identified of which 2 (n=3 and n=9) consisted of intact TEM sequences with the smaller cluster linked to an IS in ZNF274. A 99% match between 2 env from rebounding plasma RNA and this smaller cluster of intact proviral genomes was identified. Comparing proviral sequences and their IS to plasma-derived RNA sequences after an ATI reveals additional information in terms of the source of viral rebound. However, this comparison is complicated by multiple factors. For example, we found a plasma-derived RNA sequence obtained during viral rebound matched a defective proviral sequence which highlights the problem of using one HIV RNA subgenomic region for identifying replication-competent virus. In addition, ongoing viral replication during rebound may prevent a 100% match with genetically intact proviral sequences making it challenging to determine the absolute source of rebound

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, highlighting the physicochemical features and the possible pathway of formation of the particulate structure. Our findings provide a novel detailed knowledge of such a general and alternative aggregation pathway for proteins, this being crucial for a basic and broader understanding of the aggregation phenomena.This is the author's accepted manuscript and will be under embargo until the 3rd of September 2015. The final version is published by ACS in The Journal of Physical Chemistry Letters here: http://pubs.acs.org/doi/abs/10.1021/jz501614e

Bacteriophages and cryogels: A new efficient tool in bioseparation

Abstract Due to developments in the last decades in the field of protein production, new technologies for bio-separation have evolved. Especially in the field of affinity chromatography, the search for new ligands is of great interest. Besides the antibodies used as affinity ligands, synthetic ligands were introduced. Textile dyes were a first group to be introduced but despite their selectivity and capacity, the use was not very successful due to leakage and toxicity problems. More attention was focused on synthetic peptide ligands which could be synthesized using different techniques. Chemical procedures such as combinatorial chemistry, solid phase chemistry, docking simulation are applied to select peptides with affinity towards a target. By using a biological technique, Phage Display Technology, peptides can also be selected by screening peptide phage libraries towards a target. New protein production technologies have also an influence on down-stream processing techniques. Feeds such as fermentation broth, transgenic milk are particulate feed stocks which implies higher demands from the chromatographic matrices. New matrices and techniques are developed to deal with this challenge. Supermacroporous cryogel monoliths are an excellent tool for this purpose. Due to macropores with the size up to 100 µm, particulate feeds such as fermentation broth, microbial and mammalian cell cultures, milk and even whole blood can flow through the cryogel without any restriction. This thesis describes novel methods of bio-panning and as well as chromatography. Bio-panning in search for new affinity ligands is done by phage display technology. Instead of synthesizing peptides, the bacteriophages expressing a peptide on an envelope protein are used as ligands. This has several advantages compared to the pure peptide.(i) Once the phage is selected, it can be amplified in great quantities at low cost as compared to the synthesis of pure peptide ligands. (ii) By using bacteriophages a very specific ligand is available combining the effects of “affinity” and “avidity” for the optimal capture of target. By combining the cryogel monolith column with bacteriophages as affinity ligands we provided “proof of principle” for this new technique in bio-separation. Two case studies are presented. A selected peptide expressing bacteriophage clone was coupled to a cryogel monolith column. In case study one, human lactoferrin or recombinant human lactoferrin was captured from human milk or transgenic bovine milk respectively. In case study two, von Willebrand factor was captured from plasma and whole blood. In both case the captured protein of interest was recovered with a purity of >95% in a single step chromatographic procedure. Taking advantage of the structural properties of the cryogel monolith a compact bio-panning procedure, Chromato-panning, was developed. Both the screening of phage libraries as the infection of phages in E.coli cells are performed in on-column mode. As a result a significant decrease in time for bio-panning was observed. The procedure is more compact and quicker and only one panning round has to be performed in order to obtain high selective phage clones as compared to conventional bio-panning procedures

Development and screening of epoxy-spacer-phage cryogels for affinity chromatography: Enhancing the binding capacity

Macroporous epoxy cryogels can be used as an alternative for classical matrices in affinity chromatography. Due to the structural properties of cryogels, with pores of up to 100 μm, crude samples can be processed at high speed without previous manipulations such as clarification or centrifugation. Also, we previously used a peptide-expressing M13 bacteriophage as an affinity ligand. These ligands show high specificity toward the target to be purified. Combination of both, leads to a relative cost-effective one-step chromatographic set-up delivering a high purity sample (>95%), however, so far with limited capacity. To increase the binding capacity of the affinity columns, we now inserted spacers between the chromatographic matrix and the phage ligand. Both linear spacers, di-amino-alkanes (C2 -C10 ), and branched polyethyleneimine spacers with different molecular weights (800 Da-10 kDa) were analyzed. Two types of peptide expressing phage ligands, a linear 15-mer and a cyclic 6-mer, were used for screening. Up to a tenfold increase in binding capacity was observed depending on the combination of phage ligand and spacer type.status: publishe

First Report on Microcystin-LR Occurrence in Water Reservoirs of Eastern Cuba, and Environmental Trigger Factors

The factors related to cyanotoxin occurrence and its social impact, with comprehension and risk perception being the most important issues, are not yet completely understood in the Cuban context. The objectives of this research were to determine the risk extension and microcystin-LR levels, and to identify the environmental factors that trigger the toxic cyanobacteria growth and microcystin-LR occurrence in 24 water reservoirs in eastern Cuba. Samplings were performed in the early morning hours, with in situ determination and physicochemical analysis carried out in the laboratory. Microcystin-LR were determined in water and within the cells (intracellular toxins) using UPLC&ndash;MS analysis after solid phase extraction. The reservoirs studied were found to be affected by eutrophication, with high levels of TN:TP ratio and phytoplankton cell concentrations, high water temperatures and low transparency, which cause collateral effect such as cyanobacterial bloom and microcystin-LR occurrence. In Hatillo, Chal&oacute;ns, Parada, M&iacute;cara, Baragu&aacute;, Cautillo, La Yaya, Guisa and Jaibo reservoirs, concentrations of MC-LR higher than the WHO limits for drinking water (1 &micro;g&middot;L&minus;1), were detected

Apparent heterogeneity in the pIII-peptide fusion protein in single-phage clones isolated from peptide libraries

Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinities could be separated by stepwise elution. When these different fractions were re-applied, the respective differences in affinity were retained. However, when phage fractions with different apparent affinities were first amplified, an offspring was generated with again variable affinities. As the sequence of the peptide insert was the same, the heterogeneity must be ascribed to differences in avidity and although no direct evidence could be generated, we hypothesize that this is possibly due to phages displaying different numbers of the same peptide as a consequence of either proteolytic or packaging events during the amplification step in Escherichia coli

Preparation and characterization of large-format macroporous cryogel disks for use in affinity chromatography and biotechnological applications

We have prepared and evaluated larger format phage-bound epoxy-cryogel columns in order to increase the yield of bound target. Freezing thermograms showed that larger column formats (2.5-5 cm diameter) are not usable due to irregular polymerization phenomena. Preparing thin disks of 0.5 cm height with similar diameter proved to be an excellent alternative. Disks could be stacked and run in a chromatographic setup. In this way, we could increase the matrix volume, ligand-binding capacity, and finally the yield of bound target. By increasing the column volume about sevenfold, we observed a 12-fold increase of ligand density and a sevenfold increase in the yield of protein recovery in a column where phages were attached without spacer and a 10- to 34-fold increase in a spacer column, depending on the spacer used.status: publishe

Cryogel applications in microbiology.

There is a great demand for improved technologies with regard to rapid processing of nano- and microparticles. The handling of viruses in addition to microbial and mammalian cells requires the availability of appropriate adsorbents. Recent developments in macroporous gels produced at subzero temperatures (known as cryogels) have demonstrated an efficiency for processing cell and virus suspensions, cell separation and cell culture applications. Their unique combination of properties such as macroporosity, tissue-like elasticity and biocompatibility, physical and chemical stability and ease of preparation, renders these materials interesting candidates for a broad range of potential applications within microbiological research. This review describes current applications of macroporous cryogels in microbiology with a brief discussion of future perspectives