Bacterial Lysates Research Articles

Modeling the disruption of respiratory disease clinical trials by non-pharmaceutical COVID-19 interventions

Respiratory disease trials are profoundly affected by non-pharmaceutical interventions (NPIs) against COVID-19 because they perturb existing regular patterns of all seasonal viral epidemics. To address trial design with such uncertainty, we developed an epidemiological model of respiratory tract infection (RTI) coupled to a mechanistic description of viral RTI episodes. We explored the impact of reduced viral transmission (mimicking NPIs) using a virtual population and in silico trials for the bacterial lysate OM-85 as prophylaxis for RTI. Ratio-based efficacy metrics are only impacted under strict lockdown whereas absolute benefit already is with intermediate NPIs (eg. mask-wearing). Consequently, despite NPI, trials may meet their relative efficacy endpoints (provided recruitment hurdles can be overcome) but are difficult to assess with respect to clinical relevance. These results advocate to report a variety of metrics for benefit assessment, to use adaptive trial design and adapted statistical analyses. They also question eligibility criteria misaligned with the actual disease burden.

LACTIC ACID BACTERIA HYDROLYSATES AND THEIR EFFECT ON SKIN HYDRATION

Recently, interest in lactobacilli lysates is growing, and the possibilities of their use cover more and more areas of human life – medicine, immunoprophylaxis, cosmetology, food industry. The article presents a method of obtaining bacterial lysates of lactic acid bacteria, Lactobacillus genus. As a destructive agent used dry lytic enzyme preparation сytal-Rk G-10X, obtained under conditions of experimental fermentation from the culture fluid Streptomyces albus UN44. The complex lytic preparation сytal-Rk contains a group of enzymes glycosidases and peptidases, the joint action of which leads to the degradation of the cell wall of a wide range of bacteria. The evaluation of the effectiveness of the use of Cytal-Rk for the degradation of six strains of lactobacilli is presented. It is shown that the enzyme is an effective destructive agent under optimal conditions. The degree of cell degradation depended on their species, hydrolysis conditions and physical condition. Optimal conditions for obtaining hydrolysates based on native and lyophilized cells of L. delbrueckii subsp. bulgaricus LB86 was develop. When loading the microbial mass in the reaction medium – 1×109 CFU/ml (for native cells) and 1×1010 CFU/ml (for lyophilized cells) cell destruction reached almost 80% and 90%, respectively. Based on native Lactobacillus delbrueckii subsp. bulgaricus LB86 lysates were obtained and their comparative chemical analysis was performed. Sublimated cells were shown to be more sensitive to the enzyme complex and to contain more proteins and reducing sugars. Native cell lysates were more enriched in nucleic acids. Lysate of lactic acid bacteria from native cells was studied to moisturize the skin of the hands of young people aged 18–20 years. When using lysate in the cream base, it significantly increased the level of hydration of the dermis of the hands compared to the control cream base. This allows us to consider the lysate of Lactobacillus delbrueckii subsp. bulgaricus LB86 as a promising ingredient for creating cosmetics with moisturizing action.

Pre-exposure to Aerosolized Polyvalent Bacterial Lysates Protects Against Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial pneumonia of unknown cause. No therapeutic modalities can reverse or stop its ever-deteriorating course. The stimulation of lung innate immunity using bacterial lysates was found to protect against lethal pulmonary infection. Hence, this study aimed to explore whether the immune-stimulating enhancement by pretreatment with bacterial lysates led to protection against bleomycin-induced pulmonary fibrosis. C57BL/6 mice were randomly divided into 4 groups with 20 mice in each group. The mice were exposed to an aerosolized mixture of polyvalent bacterial lysates (PVBL) or phosphate-buffered saline (PBS) three times on separate days. Twenty-four hours after the last exposure, the lungs were intratracheally infused with bleomycin (BLM) or normal saline (NS). The pulmonary morphology, Ashcroft's scale of pulmonary fibrosis, and levels of pro-inflammatory cytokines such as interferon (IFN)-γ and interleukin (IL)-4 were evaluated 14 days after the intratracheal infusion. The exposure to PVBL did not induce any discernible structural abnormalities in the lungs, while the IFN-γ/IL-4 ratio increased. BLM-induced pulmonary fibrosis was associated with an overwhelming downregulation of IFN-γ and IL-4 expression. Pre-exposure to PVBL protected against BLM-induced pulmonary fibrosis, which was demonstrated by a greater reduction of Ashcroft's fibrotic score and a greater decrease in the hydroxyproline level in the lungs. Although the PVBL pre-exposure did not restore the BLM-induced downregulation of IL-4 and IFN-γ levels, the IFN-γ/IL-4 ratio was still maintained greater than the animals with BLM infusion. BLM-induced murine pulmonary fibrosis is associated with downregulation of IFN-γ and IL-4 levels. Pre-exposure to the aerosolized PVBL protects against BLM-induced pulmonary fibrosis.

Ultra-Highly Sensitive DNA Detection with Conducting Polymer-Modified Electrodes: Mechanism, Manufacture and Prospects for Rapid e-PCR

At low copy number, sequence detection by polymerase chain reaction (PCR) requires up to 30 cycles (amplification 109) to produce a reliably detectable concentration of fluorescently-labelled amplicons. The cycle number and hence detection time is determined by the analytical sensitivity of the detector. Hybridisation of complementary DNA strands to oligonucleotide-modified conducting polymer electrodes yields an increase in the charge transfer resistance for the ferri-ferrocyanide redox couple. We demonstrate sensors using screen-printed carbon electrodes modified with a conducting polymer formed from a monomer pre-functionalised with complementary oligonucleotide, with pM sensitivity for short sequences and aM for bacterial lysate, with a response time-scale of 5 min. The response is due to the variation of electrical resistance within the polymer film. We develop a mechanism based on repulsion from the solution interface of dopant anions by the charge associated with surface-bound DNA. With results for >160 single-use sensors, we formulate a response model based on percolation within a random resistor network and highlight challenges for large-scale manufacture of such sensors. Such sensors used for label-free electrochemical detection for PCR (e-PCR) would decrease the required cycle number from 30 to less than 10 and would offer a much simplified instrument construction.

Inflammatory diseases of the pharynx in children

Introduction. Inflammatory diseases of the lymphoid ring of the pharynx in children are widespread and account for more than 30% in the structure of ENT pathology. Lymphoid tissue, especially the palatine tonsils, largely determines the formation of local and general protective reactions of the child’s body.The aim of this work is to assess the efficacy and safety of using topical bacterial lysate in chronic tonsillitis of streptococcal and herpes viral etiology in frequently ill children.Materials and methods. The study was conducted on a sample of 31 children aged 7–17 who were treated in the pediatric department of the Saint Petersburg Research Institute of Ear, Throat, Nose and Speech inpatiently or outpatiently, which were divided into 2 groups. Group 1 consisted of 15 children with decompensated chronic tonsillitis of streptococcal etiology, who were prescribed tonsillectomy according to indications. Group 2 consisted of 16 children with chronic compensated tonsillitis, who, after an acute respiratory infection, showed signs of acute pharyngitis in the form of pain and discomfort in the throat, perspiration, low-grade body temperature. In both groups children were prescribed to take topical baclerial lysates.Results. A subjective pain relief of sore throat in patients (groups 1 and 2) was observed from day 2–3 of administration of the topical bacterial lysate. The clinical therapeutic effect resulted in decreased intensity of inflammation of the posterior pharyngeal wall, reduction in size and tenderness of regional lymph nodes from day 4–5 of administration of the drug. The clinical recovery from acute pharyngitis in children of both groups was observed in 98% of cases at visit 3.Conclusion. The persistence of pathogenic viruses and bacteria contributes to the development of secondary immunodeficiency states and participates in the formation of a kind of “vicious circle” In these causes prescription of topical baclerial lysates may be an effective conservation therapy as well as antibiotics and antiviral drugs. The drug showed its efficacy in the treatment of acute and chronic diseases of the pharynx in children of both viral and bacterial etiology.

Blood Bacteria-Free DNA in Septic Mice Enhances LPS-Induced Inflammation in Mice through Macrophage Response.

Although bacteria-free DNA in blood during systemic infection is mainly derived from bacterial death, translocation of the DNA from the gut into the blood circulation (gut translocation) is also possible. Hence, several mouse models with experiments on macrophages were conducted to explore the sources, influences, and impacts of bacteria-free DNA in sepsis. First, bacteria-free DNA and bacteriome in blood were demonstrated in cecal ligation and puncture (CLP) sepsis mice. Second, administration of bacterial lysate (a source of bacterial DNA) in dextran sulfate solution (DSS)-induced mucositis mice elevated blood bacteria-free DNA without bacteremia supported gut translocation of free DNA. The absence of blood bacteria-free DNA in DSS mice without bacterial lysate implies an impact of the abundance of bacterial DNA in intestinal contents on the translocation of free DNA. Third, higher serum cytokines in mice after injection of combined bacterial DNA with lipopolysaccharide (LPS), when compared to LPS injection alone, supported an influence of blood bacteria-free DNA on systemic inflammation. The synergistic effects of free DNA and LPS on macrophage pro-inflammatory responses, as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10), pro-inflammatory genes (NFκB, iNOS, and IL-1β), and profound energy alteration (enhanced glycolysis with reduced mitochondrial functions), which was neutralized by TLR-9 inhibition (chloroquine), were demonstrated. In conclusion, the presence of bacteria-free DNA in sepsis mice is partly due to gut translocation of bacteria-free DNA into the systemic circulation, which would enhance sepsis severity. Inhibition of the responses against bacterial DNA by TLR-9 inhibition could attenuate LPS-DNA synergy in macrophages and might help improve sepsis hyper-inflammation in some situations.

The first report on detecting SARS-CoV-2 inside human fecal-oral bacteria: A case series on asymptomatic family members and a child with COVID-19

Many studies report the importance of using feces as source sample for detecting SARS-CoV-2 in patients with COVID-19 symptoms but who are negative to oropharyngeal/ nasopharyngeal tests. Here, we report the case of an asymptomatic child whose family members had negative results with the rapid antigen nasopharyngeal swab tests. The 21-month-old child presented with fever, diarrhea, bilateral conjunctivitis, and conspicuous lacrimation. In this study, analysis for the presence of SARS-CoV-2 in fecal samples by using Luminex technology allowed accurate detection of the presence of the viral RNA in the feces of the child and of all her relatives, which thus resulted to be positive but asymptomatic. It is the first time that SARS-CoV-2- is observed inside human fecal-oral bacteria and outside a matrix resembling extracellular bacterial lysates, in agreement with a bacteriophage mechanism with the images obtained by transmission electron microscopy (TEM), post-embedding immunogold, and by fluorescence microscope. In addition to the typical observations of respiratory symptoms, accurate evaluation of clinical gastrointestinal and neurological symptoms, combined with efficient highly sensitive molecular testing on feces, represent an efficient approach for detecting SARS-CoV-2, and for providing the correct therapy in challenging COVID-19 cases, like the one here reported.

Diffusion NMR of alpha-synuclein in the presence of bacterial cell lysate crowders

The crowded and heterogeneous intracellular milieu has a concentration of macromolecules up to 400 mg/ml. The causes of macromolecular crowding effects are excluded volume and ‘soft' nonspecific interactions, such as hydrogen bonds, electrostatic, Van der Waals, and hydrophobic interactions. Some physiological consequences of macromolecular crowding on proteins are on protein structure, protein folding, phase separation, aggregation, and protein-protein interaction. Traditionally, macromolecular crowding effects has been studied in artificial crowders like Ficoll and Dextran.

Rapid development of a subunit nano-vaccine against drug-resistant Pseudomonas aeruginosa with effective cross-protection

The complex antigen composition and limited selection of adjuvants restricted the development of Pseudomonas aeruginosa (P. aeruginosa) subunit vaccines. The evolvement of multiple serotypes of P. aeruginosa also challenges current vaccine candidates due to their poor cross-protection capacity. Herein, we first report a facile strategy to prepare comprehensive antigens loaded nano-vaccines for the rapid development of a P. aeruginosa vaccine with a highly effective cross-protection capacity. Namely, cytosolic antigens derived from bacterial lysates (BL) of PAO1, a drug-resistant P. aeruginosa, were loaded in the mesoporous silica nanospheres (MSNs), while membrane antigens derived from double-layered membrane vesicles (DMVs) of PAO1 were further fused on MSNs surface. The engineered MSN-BL@DMV showed a clear core-shell morphology, and it significantly promoted cellular internalization and maturation of dendritic cells without additional adjuvants. After subcutaneous vaccination, MSN-BL@DMV prepared from PAO1 efficiently migrated to lymph nodes, potently induced the humoral and cellular immune responses with exquisite specificity, and substantially protected mice from the challenge of PAO1 and another drug-resistant P. aeruginosa (PA-XN-1). In summary, the MSNs-based comprehensive antigen delivery system successfully protected mice from different strains of P. aeruginosa infection, providing new insights for treatment of other refractory pathogen infections.

Corrected and Republished from: "A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against Lethal Streptococcus pneumoniae Challenge".

ABSTRACTCurrent vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells

ObjectiveMucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory...

Ефективність лікування респіраторних вірусних захворювань у дітей із використанням бактеріальних лізатів

У статті розглянуто проблему гострих респіраторних інфекційно-запальних захворювань у дітей. Описані функції системи неспецифічного захисту, надана характеристика препаратів, що застосовуються для патогенетичного лікування респіраторних інфекцій, — бактеріальних лізатів.

Бактериальные лизаты как ключевой медикаментозный компонент профилактики и лечения острых респираторных инфекций у детей

В статье описаны возможности управления системой врожденного и адаптивного иммунитета медикаментозными средствами, в частности путем использования бактериального лизата Бронхо-мунал®. Подробно представлена иммунорегулирующая роль бактериальных лизатов. Дано патогенетическое обоснование использования бактериального лизата Бронхо-мунал® для профилактики острых респираторных инфекций у детей, в том числе у часто болеющих острыми респираторными инфекциями.

Новый подход к иммунотерапии острых респираторных инфекций у детей и к профилактике бактериальных осложнений

В работе представлены механизмы действия бактериальных лизатов при острых респираторных инфекциях у детей, их значимость для активации факторов неспецифической резистентности, формирования специфического иммунитета к бактериальным агентам, особо значимым в генезе респираторных заболеваний у детей.

Acute pyelonephritis: Increased plasma membrane targeting of renal aquaporin‐2

Aquaporin-2 (AQP2) shuttling between intracellular vesicles and the apical plasma membrane is pivotal in arginine vasopressin-mediated urine concentration and is dysregulated in multiple diseases associated with water balance disorders. Children and adults with acute pyelonephritis have a urinary concentration defect and studies in children revealed increased AQP2 excretion in the urine. This study aimed to analyse AQP2 trafficking in response to acute pyelonephritis. Immunofluorescence analysis was used to evaluate subcellular localization of AQP2 and AQP2-S256A (mimicking non-phosphorylated AQP2 on serine 256) in cells stimulated with bacterial lysates and of AQP2 and pS256-AQP2 in a mouse model at day 5 of acute pyelonephritis. Western blotting was used to evaluate AQP2 levels and AQP2 phosphorylation on S256 upon incubation with bacterial lysates. Time-lapse imaging was used to measure intracellular cAMP levels in response to incubation with bacterial lysates. In cell cultures, lysates from both uropathogenic and nonpathogenic bacteria-mediated AQP2 plasma membrane targeting and increased AQP2 phosphorylation on serine 256 (pS256) without increasing cAMP levels. Both bacterial lysates induced plasma membrane targeting of AQP2-S256A. Immunofluorescence analysis of renal sections from mice after 5days of acute pyelonephritis revealed apical plasma membrane targeting of AQP2 and pS256-AQP2 in inner medullary collecting ducts. Uropathogenic bacteria induce AQP2 plasma membrane targeting in vitro and in vivo. cAMP levels were not elevated by the bacterial lysates and AQP2 plasma membrane targeting could occur without S256 phosphorylation. This may explain increased AQP2 excretion in the urine during acute pyelonephritis.

USE OF BACTERIAL LYSATE IN COMPLEX TREATMENT OF ACUTE BRONCHITIS COMBINED WITH EUTHYROID SICK SYNDROME IN PRESCHOOLERS

Objective. This study's objective was to determine bacterial lysate's effect on the state of humoral immunity (IgA, IgM, IgG) in preschool children with acute bronchitis in combination with euthyroid sick syndrome. Materials and methods. We examined 135 children of preschool age (3 to 6 years) with acute bronchitis (main group) and 28 healthy children (control group). Group Ia included 45 children with acute bronchitis who were treated according to the standard scheme. Preschoolers with acute bronchitis in Group Ib (47 patients) additionally received bacterial lysate. Group IIa included 21 children with acute bronchitis and euthyroid sick syndrome (ESS) manifestations who received standard therapy. Group IIb consisted of 22 patients with acute bronchitis and ESS who received standard therapy and were additionally given an immunomodulator. The bacterial lysate consisted of Staphylococcus aureus 6×109 colony-forming units (CFU), Streptococcus pyogenes 6×109 CFU, Streptococcus viridans 6×109 CFU, Klebsiella pneumoniae 6×109 CFU, Klebsiella ozaenae 6×109 CFU, Haemophilus influenzae B 6×109 CFU, Neisseria catarrhalis 6×109 CFU, Streptococcus pneumoniae 6×109 CFU (the latter included the following types in amount of 1×109 CFU each: TY1/EQ11, TY2/EQ22, TY3/EQ14, TY5/EQ15, TY8/EQ23, TY47/EQ24). The effectiveness of bacterial lysate in children with acute bronchitis and with or without ESS was determined by comparing the humoral immunity parameters (IgA, IgM, IgG) in the acute period (1 to 2 days after the onset) and convalescence period (7 to 10 days after the onset). Levels of immunoglobulins (IgA, IgM, and IgG) were measured by solid-phase enzyme-linked immunosorbent assay. All results were statistically processed using the SPSS 26 software. Results. No statistically significant difference was found between the values of immunoglobulins A, M, and G in children with acute bronchitis and with or without ESS manifestations in the acute period of the disease. At the same time, the above parameters were significantly higher than the corresponding values in the control group. A statistically significant difference was observed in the humoral immunity parameters of patients who were given the bacterial lysate vs. patients who were not. In children who were additionally given the bacterial lysate, IgA levels practically equaled the levels of the control group in the convalescence period. The serum levels of IgM and IgG in children who additionally received the immunomodulator tended to approach the values of the control group. Conclusions. The use of bacterial lysate positively affected the restoration of humoral homeostasis, especially on the level of immunoglobulin A, in children with acute bronchitis and with or without the euthyroid sick syndrome. The bacterial lysate demonstrated high efficiency against this disease in this group of patients.

Detecting and identifying glycoside hydrolases using cyclophellitol-derived activity-based probes.

The ability to detect active enzymes in a complex mixture of folded proteins (e.g., secretome, cell lysate) generally relies on observations of catalytic ability, necessitating the development of an activity assay that is compatible with the sample and selective for the enzyme(s) of interest. Deconvolution of the contributions of different enzymes to an observed catalytic ability further necessitates an often-challenging protein separation. The advent of broadly reactive activity-based probes (ABPs) for retaining glycoside hydrolases (GHs) has enabled an alternative, often complementary, assay for active GHs. Using activity-based protein profiling (ABPP) techniques, many retaining glycoside hydrolases can be separated, detected, and identified with high sensitivity and selectivity. This chapter outlines ABPP methods for the detection and identification of retaining glycoside hydrolases from microbial sources, including protein sample preparation from bacterial lysates and fungal secretomes, enzyme labeling and detection via fluorescence, and enzyme identification using affinity-based enrichment coupled to peptide sequencing following isobaric labeling.

In vitro anti-inflammatory and antibiofilm activities of bacterial lysates from lactobacilli against oral pathogenic bacteria.

Postbiotics are functional biological compounds, such as bacterial lysates (BLs) released from probiotic bacteria. Although postbiotics exert various bioactivities, the anti-inflammatory and antibiofilm activities of BLs against oral pathogenic bacteria have not been investigated. In the present study, pretreatment with BLs extracted from Lactobacillus plantarum and L. rhamnosus GG suppressed the mRNA and protein expression levels of inflammatory mediators induced by the lipopolysaccharide (LPS) of Porphyromonas gingivalis in RAW 264.7 cells. Both BLs attenuated P. gingivalis LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and activation of nuclear factor-κB (NF-κB), suggesting that BLs inhibit periodontal inflammatory responses by regulating the MAPK and NF-κB signaling pathways. Moreover, both BLs interfered with biofilm formation by Streptococcus mutans; however, they did not eradicate the established S. mutans biofilm. Furthermore, both BLs downregulated gtfB, gtfC, and gtfD responsible for biofilm formation by S. mutans, suggesting that BLs reduce the synthesis of extracellular polysaccharide and thereby reduce S. mutans biofilm. Taken together, these results suggest that BLs of L. plantarum and L. rhamnosus GG can attenuate periodontal inflammation and dental caries and thus contribute to the improvement of oral health.

Robotic Affinity Purification of Soluble and Insoluble Recombinant Glutathione-S-Transferase Fusion Proteins.

A completely automated purification of glutathione-S-transferase (GST) fusion proteins, either in soluble form or after renaturation of insoluble inclusion bodies, is described. Depending on the expression levels and the amount of glutathione affinity matrix employed, the protocol yields approximately 30-100μg of purified GST-fusion protein from 2mL microplate cultures. The high yield is facilitated by employing an efficient chemical/enzymatic lysis procedure for preparing bacterial cell lysates. Insoluble GST-fusion proteins are automatically refolded by a high-throughput robotic microdialysis procedure that also assesses the degree of successful refolding by integrated GST enzymatic assays and quantitation of soluble protein successfully recovered after affinity purification. For soluble GST-fusion proteins the purification procedure is normally completed within 60min, whereas urea-based denaturation-renaturation strategies typically require an additional 18h. The integration of quantitation of cell growth and affinity-purified GST-fusion protein yield allows direct comparisons of different expression constructs and the yield of soluble GST-fusion proteins to be optimized in a systematic manner.

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins.

We have developed the CL7/Im7 protein purification system to achievehigh-yield, high-purity and high-activity (HHH) products in one step. The system is based on the natural ultrahigh-affinity complex between the two small proteins encoded by colicinogenic plasmids carried by certain E. coli strains, the DNAse domain of colicin E7 (CE7; MW~15kDa) and its natural endogenous inhibitor, the immunity protein 7 (Im7; MW~10kDa). CL7 is an engineered variant of CE7, in which the toxic DNA-binding and catalytic activities have been eliminated while retaining the high affinity to Im7. CL7 is used as a protein tag, while Im7 is covalently attached to agarose beads. To make the CL7/Im7 technique easy to use, we have designed a set of the E. coli expression vectors for fusion of a target protein to the protease-cleavableCL7-tag either at the N- or the C-terminus, and also have the options of the dual (CL7/His8) tag. A subset of vectors is dedicated for cloning membrane and multisubunit proteins. The CL7/Im7 system has severalnotable advatantages over other available affinity purification techniques. First, high concentrations of the small Im7 protein are coupled to the beads resulting in the high column capacities (up to 60mg/mL). Second, an exceptional stability of Im7 allows for multiple (100+) regeneration cycles with no loss of binding capacities. Third, the CL7-tag improves protein expression levels, solubility and, in some cases, assists folding of the target proteins. Fourth, the on-column proteolytic elution produces purified proteins with few or no extra amino acid residues. Finally, the CL7/Im7 affinity is largely insensitive to high salt concentrations. For many target proteins, loading the bacterial lysates on the Im7 column in high salt is a key to high purity. Altogether, these properties of the CL7/Im7 system allow for a one-step HHH purification of most challenging, biologically and clinically significant proteins.