Mild Detergent Research Articles

Photomodulation of fatty acid composition and lipoxygenase activity in sunflower seedling cotyledons

The dynamics of fatty acid composition is an important metabolic index of seed germination and seedling development process. Present investigations indicate a preferential mobilization of fatty acids, particularly linoleic acid, out of the oil bodies in sunflower seedling cotyledons. A lack of selectivity in the utilization of saturated fatty acids during seed germination is also evident. Lipoxygenase (LOX) may play a role in the reconstitution of membrane lipid composition as cells expand during seed germination. LOX activity is absent in sunflower seeds and appears during germination. Photo modulation of LOX activity is evident by its enhanced expression in light-grown seedling cotyledons. Since sunflower LOX activity is easily solubilized by mild detergent, such as 2 % Triton X-100, the association of LOX with the membrane does not seem to be very strong unlike other membrane proteins, such as oleosins, which can be extracted only by stringent procedures. Sunflower LOX has only one pH optimum with linoleic and linolenic acids as substrates, indicating the presence of single isoform in the total soluble protein (TSP) and oil body (OB) extracts. OB-associated LOX exhibits substrate-dependent variation in its pH optima. Specific activity of TSP and OB-associated LOX increases as seedling growth progresses. Sunflower TSP-LOX activity is inhibited by some hydrophobic thiols. TSP as well as OB-associated LOX activity in sunflower cotyledons is modulated by Ca2+ availability in a negative way. A critical role of LOX is, thus, evident during seed germination in sunflower.

Structural transition in Bcl-xL and its potential association with mitochondrial calcium ion transport.

Bcl-2 family proteins are key regulators for cellular homeostasis in response to apoptotic stimuli. Bcl-xL, an antiapoptotic Bcl-2 family member, undergoes conformational transitions, which leads to two conformational states: the cytoplasmic and membrane-bound. Here we present the crystal and small-angle X-ray scattering (SAXS) structures of Bcl-xL treated with the mild detergent n-Octyl β-D-Maltoside (OM). The detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) by swapping helices α6-α8 between two monomers. Unlike Bax, a proapoptotic member of the Bcl-2 family, Bcl-xL is not converted to 3DDS homodimer upon binding BH3 peptides and ABT-737, a BH3 mimetic drug. We also designed Bcl-xL mutants which cannot dimerize and show that these mutants reduced mitochondrial calcium uptake in MEF cells. This illustrates the structural plasticity in Bcl-xL providing hints toward the probable molecular mechanism for Bcl-xL to play a regulatory role in mitochondrial calcium ion transport.

Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum

Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6–7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels. The isolated holoenzyme exhibited the subunit patterns of F-type ATP synthases containing a 5-subunit hydrophilic F1 subcomplex and a 3-subunit hydrophobic F0 subcomplex. ATP hydrolysis by the isolated HF1F0 ATP synthase was successfully detected after pretreatment with different detergents by an in-gel ATPase activity assay, which showed that the highest activity was detected in the presence of mild detergents such as LDAO; moreover, high catalytic activity in the gel was already detected after the initial incubation period of 0.5h. In contrast, HF1F0 showed extremely low ATPase activity in harsher detergents such as TODC. The isolated fully functional enzyme will form the basis for future structural studies.

Single Cell GFP-Trap Reveals Stoichiometry and Dynamics of Cytosolic Protein Complexes.

We developed in situ single cell pull-down (SiCPull) of GFP-tagged protein complexes based on micropatterned functionalized surface architectures. Cells cultured on these supports are lysed by mild detergents and protein complexes captured to the surface are probed in situ by total internal reflection fluorescence microscopy. Using SiCPull, we quantitatively mapped the lifetimes of various signal transducer and activator of transcription complexes by monitoring dissociation from the surface and defined their stoichiometry on the single molecule level.

Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers

Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here.

The active site of yeast phosphatidylinositol synthase Pis1 is facing the cytosol

Five yeast enzymes synthesizing various glycerophospholipids belong to the CDP-alcohol phosphatidyltransferase (CAPT) superfamily. They only share the so-called CAPT motif, which forms the active site of all these enzymes. Bioinformatic tools predict the CAPT motif of phosphatidylinositol synthase Pis1 as either ER luminal or cytosolic. To investigate the membrane topology of Pis1, unique cysteine residues were introduced into either native or a Cys-free form of Pis1 and their accessibility to the small, membrane permeating alkylating reagent N-ethylmaleimide (NEM) and mass tagged, non-permeating maleimides, in the presence and absence of non-denaturing detergents, was monitored. The results clearly point to a cytosolic location of the CAPT motif. Pis1 is highly sensitive to non-denaturing detergent, and low concentrations (0.05%) of dodecylmaltoside change the accessibility of single substituted Cys in the active site of an otherwise cysteine free version of Pis1. Slightly higher detergent concentrations inactivate the enzyme. Removal of the ER retrieval sequence from (wt) Pis1 enhances its activity, again suggesting an influence of the lipid environment. The central 84% of the Pis1 sequence can be aligned and fitted onto the 6 transmembrane helices of two recently crystallized archaeal members of the CAPT family. Results delineate the accessibility of different parts of Pis1 in their natural context and allow to critically evaluate the performance of different cysteine accessibility methods. Overall the results show that cytosolically made inositol and CDP-diacylglycerol can access the active site of the yeast PI synthase Pis1 from the cytosolic side and that Pis1 structure is strongly affected by mild detergents.

Effect of deoxycholic acid on Ca2+ movement, cell viability and apoptosis in human gastric cancer cells

Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca2+ movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i. DOA-evoked [Ca2+]i rises concentration dependently. The response was reduced by removing extracellular Ca2+. DOA-evoked Ca2+ entry was inhibited by store-operated Ca2+ channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca2+]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca2+]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca2+]i rises. At 100–500 μM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 μM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca2+]i rises by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via store-operated Ca2+ channels. DOA also caused Ca2+-independent apoptosis.

High-Sensitivity N-Glycoproteomic Analysis of Mouse Brain Tissue by Protein Extraction with a Mild Detergent of N-Dodecyl β-D-Maltoside

N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies.

Membrane protein expression in Lactococcus lactis.

The Gram-positive bacterium Lactococcus lactis has many properties that are ideal for the overproduction of membrane proteins in a functional form. Growth of lactococci is rapid, proceeds to high cell densities, and does not require aeration, which facilitates large-scale fermentation. The available promoter systems are strong and tightly regulated, allowing expression of toxic gene products in a controlled manner. Expressed membrane proteins are targeted exclusively to the cytoplasmic membrane, allowing the use of ionophores, ligands, and inhibitors to study activity of the membrane protein in whole cells. Constructed plasmids are stable and expression levels are highly reproducible. The relatively small genome size of the organism causes little redundancy, which facilitates complementation studies and allows for easier purification. The produced membrane proteins are often stable, as the organism has limited proteolytic capability, and they are readily solubilized from the membrane with mild detergents. Lactococci are multiple amino acid auxotrophs, allowing the incorporation of labels, such as selenomethionine. Among the few disadvantages are the low transformation frequency, AT-rich codon usage, and resistance to lysis by mechanical means, but these problems can be overcome fairly easily. We will describe in detail the protocols used to express membrane proteins in L. lactis, from cloning of the target gene to the isolation of membrane vesicles for the determination of expression levels.

Efficacy of the combined use of a facial cleanser and moisturizers for the care of mild acne patients with sensitive skin.

Acne is a common skin disease that involves the seborrheic area of the face and results from the obstruction of hair follicles followed by inflammation. Careful face washing helps to improve and prevent acne; however, intensive washing has a risk of inducing skin barrier impairment and dry skin, especially in sensitive skin. We hypothesized that skin care combining mild skin cleansing and intensive moisturizing ("combination skin care") may be effective in the care of acne in subjects with dry skin and/or sensitive skin. We developed a combination skin care with a weakly acidic foaming facial skin cleanser based on a mild detergent, an aqueous lotion with eucalyptus extract and a moisturizing gel containing pseudo-ceramide and eucalyptus extract. To optimize an ideal facial skin care system for mild acne on sensitive skin, we performed a 4-week clinical trial with 29 post-adolescent Japanese women with mild acne with dry and sensitive skin. The acne significantly decreased after this trial accompanied by the improvement of dry skin, a significantly increased endogenous ceramide level in the stratum corneum and an elongated alkyl chain length of the non-hydroxy acyl sphingosine type ceramide. No adverse events due to the test samples were observed. Based on diagnosis by a dermatologist, 97% of the subjects found the combination skin care to be "useful" or "slightly useful". Based on these findings, the combined use of a facial skin cleanser and moisturizers is safe and effective for the care of acne in post-adolescent Japanese women with sensitive skin.

Synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-β-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. (1)H NMR spectra were used to confirm a high level of deuteration in the synthesized d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

Impact of different cleaning processes on the laser damage threshold of antireflection coatings for Z-Backlighter optics at Sandia National Laboratories

We have examined how three different cleaning processes affect the laser-induced damage threshold (LIDT) of antireflection coatings for large dimension, Z-Backlighter laser optics at Sandia National Laboratories. Laser damage thresholds were measured after the coatings were created, and again 4 months later to determine which cleaning processes were most effective. Coatings that received cleaning exhibited the highest LIDTs compared to coatings that were not cleaned. In some cases, there is nearly a twofold increase in the LIDT between the cleaned and uncleaned coatings (19.4 J/cm2 compared to 39.1 J/cm2). Higher LIDTs were realized after 4 months of aging. The most effective cleaning process involved washing the coated surface with mild detergent, and then soaking the optic in a mixture of ethyl alcohol and deionized water. Also, the laser damage results indicate that the presence of nonpropagating (NP) damage sites dominates the LIDTs of almost every optic, despite the cleaning process used. NP damage sites can be attributed to defects such as nodules in the coating or surface contamination, which suggests that pursuing further improvements to the deposition or cleaning processes are worthwhile to achieve even higher LIDTs.

Impact of detergent on biophysical properties and immune response of the IpaDB fusion protein, a candidate subunit vaccine against Shigella species.

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB's cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55 °C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90 °C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine.

High molecular weight forms of mammalian respiratory chain complex II

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase.

Anti-stress proteins produced by Bacteroides thetaiotaomicron after nutrient starvation

Bacteroides thetaiotaomicron maybe one of the most adaptable intestinal bacteria due to its complex genome. Known to be an opportunistic pathogenic anaerobe, B. thetaiotaomicron has recently been described as a symbiont with anti-inflammatory properties. In this study, peptide mass finger printing technique was used to identify the stress proteins (maybe anti-stress proteins for the host) extracted from B. thetaiotaomicron grown under nutrient starvation (without heme, blood or bile) prior to be placed in an aerobic solution containing a mild non-ionic detergent derived from cholic acid. We focus here on proteins related to stress, knowing that superoxide dismutase was previously identified in the extract. In parallel, the morphology of the bacterial cells was observed using electronic microscopy before and after the extraction process. The effective antioxidant effect of the extract was evaluated in vitro against hydrogen peroxide. This work highlights the B. thetaiotaomicron ability to produce a large amount of stress proteins and to remain viable during the extraction. Budding vesicles were observed on its cell wall. The extraction process did not exceed 20 h in order to preserve the bacterial viability that decreased significantly after 24 h in preliminary studies. In our experimental conditions, an inhibitory effect of the extract was found against hydrogen peroxide. Animal models of inflammation will later check in vivo if this extract of anti-stress proteins is able to counter the respiratory burst beginning an inflammation process.

Parallel Reconstitution of Bacterial Toxins, Porins and Ion Channels into Suspended Lipid Membrane Microarrays for High-Throughput Electrophysiology

High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.

Expression, Folding, and Proton Transport Activity of Human Uncoupling Protein-1 (UCP1) in Lipid Membranes: EVIDENCE FOR ASSOCIATED FUNCTIONAL FORMS

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties

Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.

Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation.

Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ∼ 0.5-1 mg/L of E. coli culture.