Denaturing Conditions Research Articles

ИССЛЕДОВАНИЕ ВЛИЯНИЯ БЕЛКОВОГО ПРОФИЛЯ НА КРИСТАЛЛООБРАЗОВАНИЕ В МОДЕЛЬНЫХ МОЛОЧНЫХ СИСТЕМАХ С ПРОМЕЖУТОЧНОЙ ВЛАЖНОСТЬЮ

The purpose of research is to study the influence of the protein profile of model dairy systems with intermediate moisture content on the process of crystal formation of milk sugar (lactose). Research objectives: selection of formulation components for compiling a protein profile and study of its influence on the process of formation and growth of lactose crystals in model systems during storage. The objects of research were model systems with targeted adjustment of pH values in each of them, imitating skimmed condensed milk with sugar, in which the complete or partial replacement of the protein phase with whey proteins was carried out through the use of dry whey protein concentrate, dry hydrolyzed whey protein (obtained in native or denaturing conditions), dry cheese whey. Studies of microstructural changes in consistency were carried out using the method for determining the size of lactose crystals according to GOST 29245-91. It has been established that a change in the protein profile towards an increase in the content of native and transformed whey proteins in model systems led to a slowdown in the process of lactose crystal formation. At the same time, hydrolyzed whey proteins had a higher degree of crystal growth restriction. The complete replacement of the protein phase with dry whey protein concentrate and dry cheese whey contributed to the intensive growth of crystals at their initial minimum average size (relative to other samples). However, after 14 days of storage, the average crystal size in this model system was comparable to the rest of the samples (including the control), except for the sample using hydrolyzed whey proteins. Regularities of the influence of the active acidity of the medium on the size of the crystals were also revealed. A decrease in pH from 6.2 to 5.6 leads to the formation of lactose crystals of a smaller average size by 1.9–3.3 times.

Formation of Amyloid-Like Conformational States of β-Structured Membrane Proteins on the Example of the OmpF Porin from the <i>Yersinia pseudotuberculosis</i> Outer Membrane

The work presents the results of an in vitro and in silico study of the formation of amyloid-like structures under harsh denaturing conditions by the nonspecific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with a β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with an acidic pH value at elevated temperature, followed by long-term incubation at room temperature is necessary. After heating at 95 °C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of the tertiary and secondary structure of the protein, which are accompanied by an increase in the content of the total β-structure and a sharp decrease in the value of the characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to the formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of changes in intrinsic fluorescence during protein aggregation suggest that during the formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only areas with an internally disordered structure corresponding to the external loops of the porin, but also the main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.

Proximity Proteomics Reveals USP44 Forms a Complex with BRCA2 in Neuroblastoma Cells and Is Required to Prevent Chromosome Breakage.

The enzyme ubiquitin-specific protease 44 (USP44) is a deubiquitinating enzyme with identified physiological roles as a tumor suppressor and an oncogene. While some binding partners and substrates are known for USP44, the identification of other interactions may improve our understanding of its role in cancer. We therefore performed a proximity biotinylation study that identified products of several known cancer genes that are associated with USP44, including a novel interaction between BRCA2 and USP44. We expressed a fusion protein that linked USP44 and mutant Escherichia coli biotin ligase BioID in SH-SY5Y neuroblastoma cells. Control experiments were performed using BioID alone. In duplicate experiments, cells were pulsed with biotin and biotinylated proteins were isolated under denaturing conditions and the proteins were identified by mass spectrometry. The resulting list of proteins were analyzed using Enrichr and cross-referenced with the COSMIC Cancer Gene Census. We validated the association with BRCA2 using immunoprecipitation. The role of USP44 in the Fanconi anemia DNA repair pathway was investigated using chromosome analysis of wild-type or Usp44-knockout cells after exposure to mitomycin C. We identified 146 proteins that were selectively retrieved by the USP44 construct and compared with cells expressing the BioID ligase alone, including 15 gene products encoded by genes on tier 1 of the COSMIC Cancer Gene Census, including BRCA2. The association between USP44 and BRCA2 was validated through immunoprecipitation. We tested the functional role of USP44 in the Fanconi anemia DNA repair pathway through chromosome breakage analysis and found that cells lacking USP44 had a significant increase in chromosome breaks and radial chromosomes. We found that high BRCA2 transcript was correlated with poor survival in neuroblastoma, likely due to its tight association with proliferation in these tumors. Our results identified novel potential binding partners and potential substrates for USP44, including several with direct roles in cancer pathogenesis. Our results identified a novel association between BRCA2 and USP44, and a previously unknown role for USP44 in the Fanconi anemia DNA repair pathway that may contribute to its role in cancer.

Globin ferryl species: what is the nature of the protonation event at pH < 5?

The ferryl state in globins has previously been reported to undergo a protonation event below pH 5, as assessed using pH jump experiments with stopped-flow UV-Vis spectroscopy. This protonation entails hypsochromic shifts in the α and β bands (~ 20 to 40nm) and an ~ 10nm reduction in the energy difference between these two bands. We now report that in Mb this event is also characterized by a hypsochromic shift in the Soret band (~ 5nm). No similar shifts in Soret, α, and β bands are seen upon the denaturation of ferryl Mb with guanidine-suggesting that the spectroscopic changes in ferryl Mb at pH < 5 are not caused by changes in the solvent exposure or in hydrogen bonding around the ferryl unit. Under the same denaturing conditions (pH jump below pH 5, and/or guanidine), ferric-aqua and ferrous-oxy Mb show no spectral changes of the order seen in the ferryl pH jump experiments. Together, these observations suggest that the protonation event is localized on the iron-bound oxygen atom, as opposed to somewhere on a hydrogen-bonding partner. Time-dependent density functional theory (TD-DFT) calculations were not able to systematically predict the UV-Vis spectra of the heme to the level of detail needed to interpret the experimental findings in this study.

A genetically encoded anomalous SAXS ruler to probe the dimensions of intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) adopt ensembles of rapidly fluctuating heterogeneous conformations, influencing their binding capabilities and supramolecular transitions. The primary conformational descriptors for understanding IDP ensembles-the radius of gyration (RG), measured by small-angle X-ray scattering (SAXS), and the root mean square (rms) end-to-end distance (RE), probed by fluorescent resonance energy transfer (FRET)-are often reported to produce inconsistent results regarding IDP expansion as a function of denaturant concentration in the buffer. This ongoing debate surrounding the FRET-SAXS discrepancy raises questions about the overall reliability of either method for quantitatively studying IDP properties. To address this discrepancy, we introduce a genetically encoded anomalous SAXS (ASAXS) ruler, enabling simultaneous and direct measurements of RG and RE without assuming a specific structural model. This ruler utilizes a genetically encoded noncanonical amino acid with two bromine atoms, providing an anomalous X-ray scattering signal for precise distance measurements. Through this approach, we experimentally demonstrate that the ratio between RE and RG varies under different denaturing conditions, highlighting the intrinsic properties of IDPs as the primary source of the observed SAXS-FRET discrepancy rather than shortcomings in either of the two established methods. The developed genetically encoded ASAXS ruler emerges as a versatile tool for both IDPs and folded proteins, providing a unified approach for obtaining complementary and site-specific conformational information in scattering experiments, thereby contributing to a deeper understanding of protein functions.

Automated, Quantitative Capillary Western Blots to Analyze Host Cell Proteins in COVID-19 Vaccine Produced in Vero Cell Line.

Host cell protein (HCP) content is a major attribute for biological and vaccine products that must be extensively characterized prior to product licensure. Enzyme Linked Immunosorbent Assay (ELISA) and Mass Spectrometry (MS) are conventional methods for quantitative host cell protein analysis in biologic and vaccine products. Both techniques are usually very tedious, labor-intensive, and challenging to transfer to other laboratories. In addition, the ELISA methodology requires 2D SDS PAGE and 2D western blot antibody reagent validation to establish reagent coverage. This reagent coverage provides a rather weak link that is currently accepted, as the western blot is run under denaturing conditions and the ELISA is run under native conditions. Simple Western™ is a relatively new, automated, capillary western blot-based technology that allows for the separation, blotting, and detection of proteins. But, unlike traditional western blots, Simple Western™ is quantitative, allowing for the quantification of HCP content in biologic and vaccine samples. Antibody reagent validation is much more straightforward, as the reagent coverage can be directly linked between the 2D methodology and Simple Western™, as they are both run under denatured and reduced conditions. Herein we describe the development of a capillary western blot method to quantify the HCP content in samples generated using a Vero cell line for the production of an investigational live virus vaccine candidate (V590) for Coronavirus Disease-2019 (COVID-19). The HCP content in COVID-19 vaccine samples was evaluated using three methods: the new capillary western, the gold standard ELISA, and SDS-PAGE. Strong agreement was observed in the HCP content data between the capillary western and SDS PAGE methods, whereas the ELISA HCP data were outliers, suggesting that the capillary western is generating HCP concentrations closer to the true concentration. This is the first report of using capillary western technology in analyzing HCP in vaccine samples.

Characterization of the soybean oil body interface: revealing the mechanism of changes in the interfacial protein composition, structure, and function of oil bodies extracted under different denaturation conditions

We investigated the stability and rheological properties of extracts from soybean oil bodies (OB) obtained under different denaturing conditions (heating, guanidine hydrochloride, urea, and sodium dodecyl sulfate (SDS)). Differences in the interfacial and antioxidant properties exhibited by the different compositions of interfacial proteins in the extracted OB were also assessed. SDS disrupted the interfacial structure of OB, whereas the treatments under the other three denaturing conditions effectively increased the ability of the OB proteins to form a network structure. The analysis of the secondary and tertiary structures of proteins showed that all denaturation conditions affected both hydrogen bonding and hydrophobic interactions of the protein molecules, with urea leading to the most pronounced structural unfolding and rearrangement of the OB proteins. The microstructure results of the protein indicated that the protein treated with GuHCl and urea exhibited a 'shell-like' structure and structural decomposition, which can promote the pre-encapsulation of active substances through hydrogen bonding or hydrophobic interactions. The electrophoretic results further confirmed that SDS could displace 24-kDa proteins and disrupt the original membrane structure of OB, whereas urea removed most of the extrinsic proteins. The interfacial proteins extracted from OB using the four denaturing methods were different but all had enhanced interfacial and antioxidant properties compared to the control. Therefore, the extraction of OB under different denaturation conditions facilitated the hierarchical extraction of its interfacial proteins, thereby broadening its application in the food industry.

Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40kDa and ~ 35kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Inositol phosphates dynamically enhance stability, solubility, and catalytic activity of mTOR.

Mechanistic target of rapamycin (mTOR) binds the small metabolite inositol hexakisphosphate (IP6) as shown in structures of mTOR; however, it remains unclear if IP6, or any other inositol phosphate species, function as an integral structural element(s) or catalytic regulator(s) of mTOR. Here, we show that multiple, exogenously added inositol phosphate species can enhance the ability of mTOR and mechanistic target of rapmycin complex 1 (mTORC1) to phosphorylate itself and peptide substrates in invitro kinase reactions, with the higher order phosphorylated species being more potent (IP6=IP5>IP4 >> IP3). IP6 increased the VMAX and decreased the apparent KM of mTOR for ATP. Although IP6 did not affect the apparent KM of mTORC1 for ATP, monitoring kinase activity over longer reaction times showed increased product formation, suggesting inositol phosphates stabilize the active form of mTORC1 invitro. The effects of IP6 on mTOR were reversible, suggesting IP6 bound to mTOR can be exchanged dynamically with the free solvent. Interestingly, we also observed that IP6 could alter mTOR electrophoretic mobility under denaturing conditions and its solubility in the presence of manganese. Together, these data suggest for the first time that multiple inositol phosphate species (IP6, IP5, IP4, and to a lesser extent IP3) can dynamically regulate mTOR and mTORC1 by promoting a stable, more soluble active state of the kinase. Our data suggest that studies of the dynamics of inositol phosphate regulation of mTOR in cells are well justified.

Physical Isolation of Single Protein Molecules within Well‐Defined Coordination Cages to Enhance Their Stability

Encapsulation of a single protein within a confined space can lead to distinct properties compared to bulk solutions, but controlling the number of encapsulated proteins and their environment remains challenging. This study demonstrates the encapsulation of single proteins within well‐defined, tunable cavities of self‐assembled coordination cages, thereby enhancing protein stability. Within uniform cavities of size‐tunable coordination cages, 15 different proteins of varying sizes (3‐6 nm in diameter) and properties (e.g., isoelectric points and hydrophobicity) were successfully confined. Various analytical techniques confirmed that the proteins maintained their secondary structures and enzymatic activities under denaturing conditions such as exposure to organic solvents, heat, and buffers. These findings suggest that such coordination cages have the potential to serve as synthetic hosts for precisely controlling protein functions within their customizable cavities.

Physical Isolation of Single Protein Molecules within Well-Defined Coordination Cages to Enhance Their Stability.

Encapsulation of a single protein within a confined space can lead to distinct properties compared to bulk solutions, but controlling the number of encapsulated proteins and their environment remains challenging. This study demonstrates the encapsulation of single proteins within well-defined, tunable cavities of self-assembled coordination cages, thereby enhancing protein stability. Within uniform cavities of size-tunable coordination cages, 15 different proteins of varying sizes (3-6 nm in diameter) and properties (e.g., isoelectric points and hydrophobicity) were successfully confined. Various analytical techniques confirmed that the proteins maintained their secondary structures and enzymatic activities under denaturing conditions such as exposure to organic solvents, heat, and buffers. These findings suggest that such coordination cages have the potential to serve as synthetic hosts for precisely controlling protein functions within their customizable cavities.

Engineering a Mesoporous Silicon Nanoparticle Cage to Enhance Performance of a Phosphotriesterase Enzyme for Degradation of VX Nerve Agent.

The organophosphate (OP)-hydrolyzing enzyme phosphotriesterase (PTE, variant L7ep-3a) immobilized within a partially oxidized mesoporous silicon nanoparticle cage is synthesized and the catalytic performance of the enzyme@nanoparticle construct for hydrolysis of a simulant, dimethyl p-nitrophenyl phosphate (DMNP), and the live nerve agent VX is benchmarked against the free enzyme. In a neutral aqueous buffer, the optimized construct shows a ≈2-fold increase in the rate of DMNP turnover relative to the free enzyme. Enzyme@nanoparticles with more hydrophobic surface chemistry in the interior of the pores show lower catalytic activity, suggesting the importance of hydration of the pore interior on performance. The enzyme@nanoparticle construct is readily separated from the neutralized agent; the nanoparticle is found to retain DMNP hydrolysis activity through seven decontamination/recovery cycles. The nanoparticle cage stabilizes the enzyme against thermal denaturing and enzymatic (trypsin) degradation conditions relative to free enzyme. When incorporated into a topical gel formulation, the PTE-loaded nanoparticles show high activity toward the nerve agent VX in an ex vivo rabbit skin model. In vitro acetylcholinesterase (AChE) assays in human blood show that the enzyme@nanoparticle construct decontaminates VX, preserving the biological function of AChE when exposed to an otherwise incapacitating dose.

Exploring the structural heterogeneity of IgG1s: A comprehensive analysis of glycosylated and deglycosylated forms using TIMS-TOF-MS technique

Denaturation of IgG is a complex and multistep process that involves various structural changes in the protein as it loses its native conformation. IgG denaturation is crucial in therapeutic antibody development, manufacturing, and storage. Glycosylated IgG is generally more resistant to denaturing conditions due to the stabilizing effect of its glycans. In contrast, deglycosylated IgG is more vulnerable to structural disruption. In this study, we examined the intact level and large fragments of IgG glycoforms. Our investigation focused on changes in structural heterogeneity through glycosylation under denaturing conditions. Collision cross-sections (CCS) of intact level and large fragments of IgG1 molecules have been determined using reversed phase-high performance liquid chromatography-electrospray ionization-trapped ion mobility-time-of-flight mass spectrometry (RP-HPLC-ESI-TIMS-TOF-MS) technique. At the intact level, deglycosylated bevacizumab (IgG1B) and trastuzumab (IgG1T) could be differentiated at specific charge states and CCS ranges. The deglycosylated IgG1B molecule showed a broader CCS range than the IgG1T molecule. This result demonstrated that the deglycosylated IgG1T was altered from deglycosylated IgG1B in the gas phase under denaturing conditions. The lowering in the collision cross-section distribution (CCSD) values indicates a decrease in the structural heterogeneity of the intact IgG after deglycosylation. The ion mobility-mass spectrometry (IM-MS) analyses performed in this study showed that the glycans on the Fc region of IgG1s can trigger structural changes in the molecule under denaturing conditions. Depending on whether they are glycosylated or deglycosylated, various conformations of IgG1 molecules could be distinguished in RP-HPLC-TIMS-TOF-MS analysis.

Common structural features in some of the sequentially distant neurotransmitter transporters N-termini

The N-terminal regions of SLC6 transporters are sequentially unrelated, and the majority of such transporters contain only relatively short peptide N-terminal extensions. Currently, it is not clear if a diversity of N-terminal sequences represents diverse functions among the transporters or if there are common functions hidden behind similar, as yet unidentified, structures. Using alignment of amino acid sequences with the hydropathy plot, disorder prediction, and calpain recognition sites, we show that common structural features among the N-termini of some transporters might exist.We previously showed that polymeric neurotransmitter transporter N-termini exhibit very similar profiles of dynamic, time-dependent 465-595-350-750 nm absorbance metachromasia in the Bradford assay. Here we report that under certain mild denaturing conditions, filamentous aggregation of glutathione S-transferase (GST) protein results in similar near-infrared metachromasia. This effect was eliminated by further GST protein denaturation and solubilization. The results suggest that aggregation of partially denatured GST stabilizes Coomassie dye docking sites, producing a near-infrared absorbance shift similar to that observed in the polymeric unstructured N-termini of transporters.

Native Digestion and Shotgun Proteomics for Host Cell Protein Profiling of Adeno-Associated Viruses.

Host cell proteins (HCPs) are contaminants of biotherapeutics produced from engineered living systems; they can influence the product's quality, efficacy, and toxicity. Liquid chromatography coupled to mass spectrometry can detect HCPs thereby mitigating their risks. However, highly abundant biotherapeutics hamper the detection of low-level HCPs. Sample preparation termed native digestion has proven effective to preferentially digest and draw out HCPs from intact antibodies. Here, we adapted native digestion to adeno-associated viruses (AAV), which is a vector gaining popularity for gene therapy. We leveraged quantitative proteomics using capillary-flow liquid chromatography-mass spectrometry (LC-MS) and demonstrated that native digestion was more effective than applying denaturing conditions to extract the HCPs associated with different AAV serotypes.

Efficient Enzymatic Hydrolysis of the Whey Protein Fraction from Bovine Milk

Studies were conducted to optimize the hydrolysis of the major whey proteins with the serine proteases Alcalase and Protozyme, and of the immunoglobulin fraction of colostrum with Alcalase. The protein-peptide composition of the enzymatic hydrolysates of whey and colostrum was determined using high-performance liquid chromatography and electrophoresis data under denaturing conditions. The hydrolysis conditions, in conjunction with the application of a heat treatment of the substrate, were designed to enhance the efficiency of whey proteolysis using Protozyme. An increase in the proteolysis degree of the native immunoglobulin fraction of colostrum with Alcalase was also achieved. The results of the experimental work are intended for the production of a hypoallergenic protein component derived from bovine milk

Biochemically Programmable Isothermal PCR.

Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate-limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra-fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection.

Biochemical Properties of CARM1: Impact on Western Blotting and Proteomic Studies.

CARM1 is an arginine methyltransferase that has crucial roles in a number of cellular pathways and is being explored as a therapeutic target in diseases such as cancer and neurodegenerative disorders. Its deregulation at the protein level was found to have potential prognostic value, and as such, its protein levels are regularly assessed through the common practice of western blotting (WB). Our group uncovered that CARM1 has biochemical properties that complicate its analysis by standard WB sample preparation techniques. Here, we show that CARM1 has the ability to form SDS-resistant aggregates that effectively hinder gel migration in SDS-PAGE. CARM1 levels and the temperature at the denaturation step can both influence CARM1 aggregation, which prompts the use of additional measures to ensure representative detection at the protein level. We have demonstrated the formation of CARM1 aggregates in both cell and tissue extracts, making these findings an important consideration for any CARM1-related study. We also show how aggregate formation in models of CARM1 overexpression can hinder proteomic studies. Having identified factors that can induce CARM1 aggregation, we suggest alternative sample preparation techniques that allow for clear resolution of the protein in stringent denaturing conditions while avoiding aggregation.

Efficient Production and Purification of Bioactive E50-52-ClassIIa Peptidic Bacteriocin Is Achieved through Fusion with the Catalytic Domain of Lysostaphin-Class III Bacteriocin.

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium, has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-classIII bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli, but it became soluble using phosphate buffer (pH7.4) supplemented with 8M urea. Purification using immobilized-Ni2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with &gt;95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori, a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100mg per liter of culture. To thebest of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H.pylori. This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.

DNA Extraction and PCR Optimization of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner

DNA isolation is an important step for further molecular analysis. Coffee plants contain polysaccharides, polyphenols, and secondary metabolites which can contaminate the results of DNA isolation. This study was conducted to isolate DNA from arabica and robusta coffee leaves using a modified CTAB method and PCR optimization for amplification of matK and rbcL genes. DNA was isolated using buffers (CTAB 10%, PVP 1%, β-mercaptoethanol 1%, Tris-HCl 1M, NaCl 5M, and EDTA 0.5%), and eluted with TE-RNase. Genomic DNA of ten coffee plants was successfully isolated with concentrations ranging from 33-146 nm/µL with purity (A260/A280) 1.7-1.9. Based on the genomic DNA isolated, matK and rbcL genes were amplified with initial denaturation conditions of 94°C (for 1 minute of matK and 4 minutes of rbcL) followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing (adjusted to each primer), and extension at 72°C for 1 minute. The optimal PCR conditions were effective to amplify matK (900 bp) and rbcL (600 bp) genes. Thus, the modified DNA isolation method and PCR optimization can be used as an efficient tool for further molecular analysis of Coffea arabica L. and Coffea canephora Pierre ex A. Froehner.