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  1. A study of the haemolysin test in the identification of V. Cholerae biotype, El Tor
    Subrahmanyam C
    Med J Malaya, 1966 Mar;20(3):234-9.
    PMID: 4223073
    Matched MeSH terms: Hemolysin Proteins/pharmacology*
  2. Molecular tissue profiling by MALDI imaging: recent progress and applications in cancer research
    Lee PY, Yeoh Y, Omar N, Pung YF, Lim LC, Low TY
    Crit Rev Clin Lab Sci, 2021 11;58(7):513-529.
    PMID: 34615421 DOI: 10.1080/10408363.2021.1942781
    Matrix-assisted laser desorption/ionization (MALDI) imaging is an emergent technology that has been increasingly adopted in cancer research. MALDI imaging is capable of providing global molecular mapping of the abundance and spatial information of biomolecules directly in the tissues without labeling. It enables the characterization of a wide spectrum of analytes, including proteins, peptides, glycans, lipids, drugs, and metabolites and is well suited for both discovery and targeted analysis. An advantage of MALDI imaging is that it maintains tissue integrity, which allows correlation with histological features. It has proven to be a valuable tool for probing tumor heterogeneity and has been increasingly applied to interrogate molecular events associated with cancer. It provides unique insights into both the molecular content and spatial details that are not accessible by other techniques, and it has allowed considerable progress in the field of cancer research. In this review, we first provide an overview of the MALDI imaging workflow and approach. We then highlight some useful applications in various niches of cancer research, followed by a discussion of the challenges, recent developments and future prospect of this technique in the field.
    Matched MeSH terms: Proteins*
  3. Fulltext Cell-Based High-Throughput Screening Protocol for Discovering Antiviral Inhibitors Against SARS-COV-2 Main Protease (3CLpro)
    Rothan HA, Teoh TC
    Mol Biotechnol, 2021 Mar;63(3):240-248.
    PMID: 33464543 DOI: 10.1007/s12033-021-00299-7
    The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.
    Matched MeSH terms: Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Recombinant Fusion Proteins/chemistry; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Green Fluorescent Proteins/chemistry
  4. Acetogenins Exhibit Potential BCL-XL Inhibitor for the Induction of Apoptosis in the Molecular Docking Study
    Nordin N, Khimani K, Abd Ghani MF
    Curr Drug Discov Technol, 2021;18(6):e010921191171.
    PMID: 33563198 DOI: 10.2174/1570163818666210204202426
    BACKGROUND: Anti-apoptotic protein BCL-XL plays a vital role in tumorigenesis and cancer chemotherapy resistance, resulting in a good target for cancer treatment. Understanding the function of BCL-XL has driven the progression of a new class of cancer drugs that can mimic its natural inhibitors, BH3-only proteins, to trigger apoptosis. This mimicking is initiated through acetogenins due to their excellent biological properties. Acetogenins, which can be isolated from Annonaceae plants, have a unique structure along with several oxygenated functionalities.

    OBJECTIVE: Based on their biological capability, various acetogenins were studied in the present study and compared alongside ABT-737 on molecular docking.

    METHODS: The docking simulation of acetogenins was performed using AutoDock Vina software.

    RESULTS: Our findings have shown eleven acetogenins-BCL-XL protein complex, namely, muricin B (2), muricin F (4), muricin H (6), muricin I (7), xylomaticin (9), annomontacin (12), annonacin (14), squamocin (15), squamostatin A (16), bullatacin (20) and annoreticulin (21) exhibited strong binding affinities lower than - 10.4 kcalmol-1 as compared to ABT-373-BCL-XL complex. Six hydrogen bonds along with hydrophobic interaction were detected on the complex of BCL-XL with muricin B (2), muricin G (5), corossolone (11), and isoannonacin-10-one A (18).

    CONCLUSION: These findings indicated that some acetogenins could represent a new potential BCLXL inhibitor that could mimic the BH3-only protein for the induction of apoptosis in cancer chemotherapy.

    Matched MeSH terms: Apoptosis Regulatory Proteins/pharmacology
  5. Flotation removal of the microalga Nannochloropsis sp. using Moringa protein-oil emulsion: A novel green approach
    Kandasamy G, Shaleh SRM
    Bioresour Technol, 2018 Jan;247:327-331.
    PMID: 28950142 DOI: 10.1016/j.biortech.2017.08.187
    A new approach to recover microalgae from aqueous medium using a bio-flotation method is reported. The method involves utilizing a Moringa protein extract - oil emulsion (MPOE) for flotation removal of Nannochloropsis sp. The effect of various factors has been assessed using this method, including operating parameters such as pH, MPOE dose, algae concentration and mixing time. A maximum flotation efficiency of 86.5% was achieved without changing the pH condition of algal medium. Moreover, zeta potential analysis showed a marked difference in the zeta potential values when increase the MPOE dose concentration. An optimum condition of MPOE dosage of 50ml/L, pH 8, mixing time 4min, and a flotation efficiency of greater than 86% was accomplished. The morphology of algal flocs produced by protein-oil emulsion flocculant were characterized by microscopy. This flotation method is not only simple, but also an efficient method for harvesting microalgae from culture medium.
    Matched MeSH terms: Proteins*
  6. Dietary protein interacts with polygenic risk scores and modulates serum concentrations of C-reactive protein in overweight and obese Malaysian adults
    Tan PY, Amini F, Mitra SR
    Nutr Res, 2022 Nov;107:75-85.
    PMID: 36206635 DOI: 10.1016/j.nutres.2022.09.002
    Dietary intake may interact with gene variants and modulate inflammatory status. This study aimed to investigate the combined effect of fat mass and obesity-associated rs9930501, rs9930506, and rs9932754 and beta-2 adrenergic receptor rs1042713 on C-reactive protein (CRP) concentrations using polygenic risk scores (PRS), and modulatory effect of dietary nutrients on these associations. We hypothesized that higher protein intake is associated with lower inflammatory status in individuals genetically predisposed to obesity. PRS was computed as the weighted sum of the risk alleles possessed and stratified into first (0-0.64), second (0.65-3.59), and third (3.60-8.18) tertiles. A total of 128 overweight and obese Malaysian adults were dichotomized into groups of low and elevated inflammatory status (CRP concentrations ≤3 and >3 mg/L, respectively). One-half of the study participants (51%) were found to have elevated inflammatory status. Second- and third-tertile PRS were significantly associated with increased odds of elevated inflammatory status, 7.56 (95% confidence interval [CI], 1.98-28.80; adjusted P = .003) and 3.87 (95% CI, 1.10-13.60; adjusted P = .035), respectively. Individuals in the third-tertile PRS had significantly lower CRP concentrations (4.61 ± 1.3 mg/L vs 9.60 ± 2.6 mg/L, P = .019) when consuming ≥14% energy from protein (with an average of 18.0% ± 2.4%, 43.0% ± 7.7%, and 39.0% ± 8.0% energy from protein, carbohydrate, and fat per day). In conclusion, third-tertile PRS was significantly associated with increased odds of elevated CRP; higher protein intake may alleviate inflammatory status and reduce CRP concentrations systemically in those individuals.
    Matched MeSH terms: Dietary Proteins*
  7. Fulltext Impact of cavitation on the structure and functional quality of extracted protein from food sources - An overview
    Kamal H, Ali A, Manickam S, Le CF
    Food Chem, 2023 May 01;407:135071.
    PMID: 36493478 DOI: 10.1016/j.foodchem.2022.135071
    Increasing protein demands directly require additional resources to those presently and recurrently available. Emerging green technologies have witnessed an escalating interest in "Cavitation Processing" (CP) to ensure a non-invasive, non-ionizing and non-polluting extraction. The main intent of this review is to present an integrated summary of cavitation extraction methods specifically applied to food protein sources. Along with a comparative assessment carried out for each type of cavitation model, protein extraction yield and implications on the extracted protein's structural and functional properties. The basic principle of cavitation is due to the pressure shift in the liquid flow within milliseconds. Hence, cavitation emerges similar to boiling; however, unlike boiling (temperature change), cavitation occurs due to pressure change. Characterization and classification of sample type is also a prime candidate when considering the applications of cavitation models in food processing. Generally, acoustic and hydrodynamic cavitation is applied in food applications including extraction, brewing, microbial cell disruption, dairy processing, emulsification, fermentation, waste processing, crystallisation, mass transfer and production of bioactive peptides. Micro structural studies indicate that shear stress causes disintegration of hydrogen bonds and Van der Waals interactions result in the unfolding of the protein's secondary and/or tertiary structures. A change in the structure is not targeted but rather holistic and affects the physicochemical, functional, and nutritional properties. Cavitation assisted extraction of protein is typically studied at a laboratory scale. This highlights limitations against the application at an industrial scale to obtain potential commercial gains.
    Matched MeSH terms: Proteins*
  8. Microalgal extract as bio-coating to enhance biofilm growth of marine microalgae on microporous membranes
    Tong CY, Chua MX, Tan WH, Derek CJC
    Chemosphere, 2023 Feb;315:137712.
    PMID: 36592830 DOI: 10.1016/j.chemosphere.2022.137712
    Microalgal biofilm is a popular platform for algal production, nutrient removal and carbon capture; however, it suffers from significant biofilm exfoliation under shear force exposure. Hence, a biologically-safe coating made up of algal extracellular polymeric substances (EPS) was utilized to secure the biofilm cell retention and cell loading on commercial microporous membrane (polyvinylidene fluoride), making the surfaces more hydrophobic (contact angle increase up to 12°). Results demonstrated that initial cell adhesion of three marine microalgae (Amphora coffeaeformis, Cylindrotheca fusiformis and Navicula incerta) was enhanced by at least 1.3 times higher than that of pristine control within only seven days with minimized biofilm exfoliation issue due to uniform distribution of sticky transparent exopolymer particles. Bounded extracellular polysaccharide gathered was approximately 23% higher on EPS-coated membranes to improve the biofilm's hydraulic resistance, whereas bounded extracellular protein would only be substantially elevated after the attached cells re-accommodate themselves onto the EPS pre-coating of themselves. In accounting the rises of hydrophobic protein content, biofilm was believed to be more stabilized, presumably via hydrophobic interactions. EPS biocoating would generate a groundswell of interest for bioprocess intensifications though there are lots of inherent technical and molecular challenges to be further investigated in future.
    Matched MeSH terms: Proteins/metabolism
  9. Smart systems in producing algae-based protein to improve functional food ingredients industries
    Neo YT, Chia WY, Lim SS, Ngan CL, Kurniawan TA, Chew KW
    Food Res Int, 2023 Mar;165:112480.
    PMID: 36869493 DOI: 10.1016/j.foodres.2023.112480
    Production and extraction systems of algal protein and handling process of functional food ingredients need to control several parameters such as temperature, pH, intensity, and turbidity. Many researchers have investigated the Internet of Things (IoT) approach for enhancing the yield of microalgae biomass and machine learning for identifying and classifying microalgae. However, there have been few specific studies on using IoT and artificial intelligence (AI) for production and extraction of algal protein as well as functional food ingredients processing. In order to improve the production of algal protein and functional food ingredients, the implementation of smart system is a must to have real-time monitoring, remote control system, quick response to sudden events, prediction and characterisation. Techniques of IoT and AI are expected to help functional food industries to have a big breakthrough in the future. Manufacturing and implementation of beneficial smart systems are important to provide convenience and to increase the efficiency of work by using the interconnectivity of IoT devices to have good capturing, processing, archiving, analyzing, and automation. This review investigates the possibilities of implementation of IoT and AI in production and extraction of algal protein and processing of functional food ingredients.
    Matched MeSH terms: Algal Proteins*
  10. Ultrasound-assisted phosphorylation of goose myofibrillar proteins: improving protein structure and functional properties
    Zhou Y, Sun Y, Pan D, Xia Q, Zhou C
    J Sci Food Agric, 2023 Aug 30;103(11):5412-5421.
    PMID: 37038882 DOI: 10.1002/jsfa.12616
    BACKGROUND: Goose meat is rough and embedded with dense connective tissue, impairing protein solubility. Therefore, to improve the functional properties of goose myofibrillar protein (GMP), ultrasound was used to assist the phosphorylation of GMP.

    RESULTS: The fact that GMP attached covalently with the phosphate group of sodium tripolyphosphate (GMP-STP) was disclosed directly by Fourier transform infrared spectroscopy. Furthermore, ultrasound significantly improved the hydrophobicity and solubility of GMP-STP, which could be attributed to the conversion of α-helix to β-sheet, β-turns, and random coils by sonication. The spatial stabilization of the protein phosphorylation process was boosted by ultrasound, making the droplets more dispersed, and thus an improvement in the functional properties of GMP-STP was observed. Water-holding capacity, oil-binding capacity, and emulsifying and foaming properties were best at an ultrasound power of 400 W.

    CONCLUSION: Ultrasound-assisted phosphorylation has great potential to modulate the structure-function relationship of proteins. © 2023 Society of Chemical Industry.

    Matched MeSH terms: Proteins*
  11. A potent anticoagulant hybrid of snake venom derived FIX-binding protein and anti-factor IX RNA aptamer: Assessed by in-silico and electrochemical analyses
    Krishnan H, Gopinath SCB
    Int J Biol Macromol, 2023 Aug 30;247:125740.
    PMID: 37423441 DOI: 10.1016/j.ijbiomac.2023.125740
    Anticoagulant therapies are crucial in the management of surgical complications as well as the prophylaxis of thrombosis. Many studies are being conducted on the Habu snake-venom anticoagulant, FIX-binding protein (FIX-Bp), for its greater potency and strong affinity to FIX clotting factor. On the other hand, the capacity to promptly reverse such acute anticoagulation is equally important. Combining a reversible anticoagulant with FIX-Bp may be advantageous in maintaining the balance between adequate anticoagulation and repealing when necessary. In this study, authors integrated FIX-Bp and RNA aptamer-based anticoagulants into a single target, FIX clotting factor, in order to achieve a robust anticoagulant effect. An in-silico and electrochemical approach were used to investigate the combination of FIX-Bp and RNA aptamers as a bivalent anticoagulant and to verify the competing or predominant binding sites of each anticoagulant. The in-silico analysis discovered that both the venom- and aptamer-anticoagulant had a strong affinity for the FIX protein at the Gla-domain and EGF-1 domain by holding 9 conventional hydrogen bonds with the binding energy of -34.859 kcal/mol. The electrochemical technique verified that both anticoagulants had different binding sites. The impedance load upon RNA aptamer binding to FIX protein was 14 %, whereas the addition of FIX-Bp caused a significant impedance rise of 37 %. This indicates that the addition of aptamers prior to FIX-Bp is a promising strategy for the conception of a hybrid anticoagulant.
    Matched MeSH terms: Carrier Proteins/chemistry
  12. Recent Advances in Overexpression of Functional Recombinant Lipases
    Alias FL, Nezhad NG, Normi YM, Ali MSM, Budiman C, Leow TC
    Mol Biotechnol, 2023 Nov;65(11):1737-1749.
    PMID: 36971996 DOI: 10.1007/s12033-023-00725-y
    Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
    Matched MeSH terms: Recombinant Proteins/chemistry
  13. Fulltext Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the histone-like HU protein from Spiroplasma melliferum KC3
    Boyko K, Gorbacheva M, Rakitina T, Korzhenevskiy D, Vanyushkina A, Kamashev D, et al.
    Acta Crystallogr F Struct Biol Commun, 2015 Jan 01;71(Pt 1):24-7.
    PMID: 25615963 DOI: 10.1107/S2053230X14025333
    HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed in space group C2 and the structure of the protein was solved by the molecular-replacement method with one monomer per asymmetric unit.
    Matched MeSH terms: Bacterial Proteins/biosynthesis; Bacterial Proteins/isolation & purification; Bacterial Proteins/chemistry*; DNA-Binding Proteins/biosynthesis; DNA-Binding Proteins/isolation & purification; DNA-Binding Proteins/chemistry*
  14. Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system
    Eskandari A, Nezhad NG, Leow TC, Rahman MBA, Oslan SN
    World J Microbiol Biotechnol, 2023 Dec 08;40(1):39.
    PMID: 38062216 DOI: 10.1007/s11274-023-03851-6
    Yeasts serve as exceptional hosts in the manufacturing of functional protein engineering and possess industrial or medical utilities. Considerable focus has been directed towards yeast owing to its inherent benefits and recent advancements in this particular cellular host. The Pichia pastoris expression system is widely recognized as a prominent and widely accepted instrument in molecular biology for the purpose of generating recombinant proteins. The advantages of utilizing the P. pastoris system for protein production encompass the proper folding process occurring within the endoplasmic reticulum (ER), as well as the subsequent secretion mediated by Kex2 as a signal peptidase, ultimately leading to the release of recombinant proteins into the extracellular environment of the cell. In addition, within the P. pastoris expression system, the ease of purifying recombinant protein arises from its restricted synthesis of endogenous secretory proteins. Despite its achievements, scientists often encounter persistent challenges when attempting to utilize yeast for the production of recombinant proteins. This review is dedicated to discussing the current achievements in the usage of P. pastoris as an expression host. Furthermore, it sheds light on the strategies employed in the expression system and the optimization and development of the fermentative process of this yeast. Finally, the impediments (such as identifying high expression strains, improving secretion efficiency, and decreasing hyperglycosylation) and successful resolution of certain difficulties are put forth and deliberated upon in order to assist and promote the expression of complex proteins in this prevalent recombinant host.
    Matched MeSH terms: Recombinant Proteins/metabolism
  15. Fulltext Monoallelic variants resulting in substitutions of MAB21L1 Arg51 Cause Aniridia and microphthalmia
    Hall HN, Bengani H, Hufnagel RB, Damante G, Ansari M, Marsh JA, et al.
    PLoS One, 2022;17(11):e0268149.
    PMID: 36413568 DOI: 10.1371/journal.pone.0268149
    Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.
    Matched MeSH terms: Eye Proteins/genetics; RNA-Binding Proteins/genetics; Homeodomain Proteins/genetics; Intracellular Signaling Peptides and Proteins/genetics
  16. Essential factors, advanced strategies, challenges, and approaches involved for efficient expression of recombinant proteins in Escherichia coli
    Eskandari A, Nezhad NG, Leow TC, Rahman MBA, Oslan SN
    Arch Microbiol, 2024 Mar 12;206(4):152.
    PMID: 38472371 DOI: 10.1007/s00203-024-03871-2
    Producing recombinant proteins is a major accomplishment of biotechnology in the past century. Heterologous hosts, either eukaryotic or prokaryotic, are used for the production of these proteins. The utilization of microbial host systems continues to dominate as the most efficient and affordable method for biotherapeutics and food industry productions. Hence, it is crucial to analyze the limitations and advantages of microbial hosts to enhance the efficient production of recombinant proteins on a large scale. E. coli is widely used as a host for the production of recombinant proteins. Researchers have identified certain obstacles with this host, and given the growing demand for recombinant protein production, there is an immediate requirement to enhance this host. The following review discusses the elements contributing to the manifestation of recombinant protein. Subsequently, it sheds light on innovative approaches aimed at improving the expression of recombinant protein. Lastly, it delves into the obstacles and optimization methods associated with translation, mentioning both cis-optimization and trans-optimization, producing soluble recombinant protein, and engineering the metal ion transportation. In this context, a comprehensive description of the distinct features will be provided, and this knowledge could potentially enhance the expression of recombinant proteins in E. coli.
    Matched MeSH terms: Recombinant Proteins/metabolism
  17. Selection and characterization of ssDNA aptamer targeting Macrobrachium rosenbergii nodavirus capsid protein: A potential capture agent in gold-nanoparticle-based aptasensor for viral protein detection
    Ghadin N, Yusof NAM, Syarul Nataqain B, Raston NHA, Low CF
    J Fish Dis, 2024 Feb;47(2):e13892.
    PMID: 38014615 DOI: 10.1111/jfd.13892
    The giant freshwater prawn holds a significant position as a valuable crustacean species cultivated in the aquaculture industry, particularly well-known and demanded among the Southeast Asian countries. Aquaculture production of this species has been impacted by Macrobrachium rosenbergii nodavirus (MrNV) infection, which particularly affects the larvae and post-larvae stages of the prawn. The infection has been recorded to cause mortality rates of up to 100% among the affected prawns. A simple, fast, and easy to deploy on-site detection or diagnostic method is crucial for early detection of MrNV to control the disease outbreak. In the present study, novel single-stranded DNA aptamers targeting the MrNV capsid protein were identified using the systematic evolution of ligands by exponential enrichment (SELEX) approach. The aptamer was then conjugated with the citrate-capped gold nanoparticles (AuNPs), and the sensitivity of this AuNP-based aptasensor for the detection of MrNV capsid protein was evaluated. Findings revealed that the aptamer candidate, APT-MrNV-CP-1 was enriched throughout the SELEX cycle 4, 9, and 12 with the sequence percentage of 1.76%, 9.09%, and 12.42%, respectively. The conjugation of APT-MrNV-CP-1 with citrate-capped AuNPs exhibited the highest sensitivity in detecting the MrNV capsid protein, where the presence of 62.5 nM of the viral capsid protein led to a significant agglomeration of the AuNPs. This study demonstrated the practicality of an AuNP-based aptasensor for disease diagnosis, particularly for detecting MrNV infection in giant freshwater prawns.
    Matched MeSH terms: Viral Proteins/genetics; Capsid Proteins/genetics; Capsid Proteins/metabolism
  18. Functional characterisation of cellobiohydrolase I (Cbh1) from Trichoderma virens UKM1 expressed in Aspergillus niger
    Wahab AFFA, Abdul Karim NA, Ling JG, Hasan NS, Yong HY, Bharudin I, et al.
    Protein Expr Purif, 2019 02;154:52-61.
    PMID: 30261309 DOI: 10.1016/j.pep.2018.09.014
    Cellobiohydrolases catalyze the processive hydrolysis of cellulose into cellobiose. Here, a Trichoderma virens cDNA predicted to encode for cellobiohydrolase (cbhI) was cloned and expressed heterologously in Aspergillus niger. The cbhI gene has an open reading frame of 1518 bp, encoding for a putative protein of 505 amino acid residues with a calculated molecular mass of approximately 54 kDa. The predicted CbhI amino acid sequence has a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region and showed high sequence homology with glycoside hydrolase family 7 proteins. The partially purified enzyme has an optimum pH of 4.0 with stability ranging from pH 3.0 to 6.0 and an optimum temperature of 60 °C. The partially purified CbhI has a specific activity of 4.195 Umg-1 and a low Km value of 1.88 mM when p-nitrophenyl-β-D-cellobioside (pNPC) is used as the substrate. The catalytic efficiency (kcat/Km) was 5.68 × 10-4 mM-1s-1, which is comparable to the CbhI enzymes from Trichoderma viridae and Phanaerochaete chrysosporium. CbhI also showed activity towards complex substrates such as Avicel (0.011 Umg-1), which could be useful in complex biomass degradation. Interestingly, CbhI also exhibited a relatively high inhibition constant (Ki) for cellobiose with a value of 8.65 mM, making this enzyme more resistant to end-product inhibition compared to other fungal cellobiohydrolases.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/chemistry
  19. Purification of BmR1 recombinant protein
    Arifin N, Basuni M, Lan CA, Yahya AR, Noordin R
    Protein J, 2010 Oct;29(7):509-15.
    PMID: 20845068 DOI: 10.1007/s10930-010-9281-1
    This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry; Helminth Proteins/genetics; Helminth Proteins/isolation & purification*; Helminth Proteins/metabolism; Helminth Proteins/chemistry
  20. A new cold-adapted, organic solvent stable lipase from mesophilic Staphylococcus epidermidis AT2
    Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Protein J, 2014 Jun;33(3):296-307.
    PMID: 24777627 DOI: 10.1007/s10930-014-9560-3
    The gene encoding a cold-adapted, organic solvent stable lipase from a local soil-isolate, mesophilic Staphylococcus epidermidis AT2 was expressed in a prokaryotic system. A two-step purification of AT2 lipase was achieved using butyl sepharose and DEAE sepharose column chromatography. The final recovery and purification fold were 47.09 % and 3.45, respectively. The molecular mass of the purified lipase was estimated to be 43 kDa. AT2 lipase was found to be optimally active at pH 8 and stable at pH 6-9. Interestingly, this enzyme demonstrated remarkable stability at cold temperature (<30 °C) and exhibited optimal activity at a temperature of 25 °C. A significant enhancement of the lipolytic activity was observed in the presence of Ca(2+), Tween 60 and Tween 80. Phenylmethylsulfonylfluoride, a well known serine inhibitor did not cause complete inhibition of the enzymatic activity. AT2 lipase exhibited excellent preferences towards long chain triglycerides and natural oils. The lipolytic activity was stimulated by dimethylsulfoxide and diethyl ether, while more than 50 % of its activity was retained in methanol, ethanol, acetone, toluene, and n-hexane. Taken together, AT2 lipase revealed highly attractive biochemical properties especially because of its stability at low temperature and in organic solvents.
    Matched MeSH terms: Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Bacterial Proteins/chemistry*; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry*
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