Displaying publications 1 - 20 of 360 in total

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  1. Manuvera VA, Kharlampieva DD, Bobrovsky PA, Grafskaia EN, Brovina KA, Lazarev VN
    Biochem Biophys Res Commun, 2024 Feb 12;696:149473.
    PMID: 38241814 DOI: 10.1016/j.bbrc.2024.149473
    The saliva of the medicinal leech contains various anticoagulants. Some of them, such as hirudin, are well known. However, it is reasonable to believe that not all anticoagulant proteins from medicinal leech saliva have been identified. We previously performed a comprehensive study of the transcriptome, genome, and proteome of leech salivary gland cells, which led to the discovery of several previously unknown hypothetical proteins that may have anticoagulant properties. Subsequently, we obtained a series of recombinant proteins and investigated their impact on coagulation in in vitro assays. We identified a previously undescribed protein that exhibited a high ability to suppress coagulation. The His-tagged recombinant protein was expressed in Escherichia coli and purified using metal chelate chromatography. To determine its activity, commonly used coagulation methods were used: activated partial thromboplastin time, prothrombin time, and thrombin inhibition clotting assay. Clotting and chromogenic assays for factor Xa inhibition were performed to evaluate anti-Xa activity. We used recombinant hirudin as a control anticoagulant protein in all experiments. The new protein showed significantly greater inhibition of coagulation than hirudin at the same molar concentrations in the activated partial thrombin time assay. However, hirudin demonstrated better results in the direct thrombin inhibition test, although the tested protein also exhibited the ability to inhibit thrombin. The chromogenic analysis of factor Xa inhibition revealed no activity, whereas the clotting test for factor Xa showed the opposite result. Thus, a new powerful anticoagulant protein has been discovered in the medicinal leech. This protein is homologous to antistatin, with 28 % identical amino acid residues. The recombinant protein was expressed in E. coli. This protein is capable of directly inhibiting thrombin, and based on indirect evidence, other proteases of the blood coagulation cascade have been identified.
    Matched MeSH terms: Recombinant Proteins/metabolism
  2. Che Hussian CHA, Leong WY
    Prep Biochem Biotechnol, 2024 Feb;54(2):150-158.
    PMID: 37233514 DOI: 10.1080/10826068.2023.2217507
    Therapeutic proteins are recombinant proteins generated through recombinant DNA technology and have attracted a great deal of interest in numerous applications, including pharmaceutical, cosmetic, human and animal health, agriculture, food, and bioremediation. Producing therapeutic proteins on a large scale, mainly in the pharmaceutical industry, necessitates a cost-effective, straightforward, and adequate manufacturing process. In industry, a protein separation technique based mainly on protein characteristics and modes of chromatography will be applied to optimize the purification process. Typically, the downstream process of biopharmaceutical operations may involve multiple chromatography phases that require the use of large columns pre-packed with resins that must be inspected before use. Approximately 20% of the proteins are assumed to be lost at each purification stage during the production of biotherapeutic products. Hence, to produce a high quality product, particularly in the pharmaceutical industry, the correct approach and understanding of the factors influencing purity and yield during purification are necessary.
    Matched MeSH terms: Recombinant Proteins/metabolism
  3. Morozova OV, Manuvera VA, Barinov NA, Subcheva EN, Laktyushkin VS, Ivanov DA, et al.
    Arch Biochem Biophys, 2024 Feb;752:109843.
    PMID: 38072298 DOI: 10.1016/j.abb.2023.109843
    Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for β-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.
    Matched MeSH terms: Recombinant Proteins
  4. 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
  5. Goa Y, Du JG, Jirapattharasate C, Galon E, Ji SW, Ran ZG, et al.
    Trop Biomed, 2023 Dec 01;40(4):400-405.
    PMID: 38308826 DOI: 10.47665/tb.40.4.004
    Beta toxin (CPB) is a lethal toxin and plays a key role in enterotoxemia of ruminants caused by Clostridium perfringens type C strain. The existing vaccines based on crude CPB need time-consuming detoxification and difficult quality control steps. In this study, we synthesized the rCPBm4 of C. perfringens type C strain and small ubiquitin-like modifier (SUMO)-tag CPBm4 (rSUMO-CPBm4) by introducing four amino acid substitutions: R212E, Y266A, L268G, and W275A. Compared with rCPBm4, rSUMO-CPBm4 was expressed with higher solubility in Escherichia coli BL21 (DE3). Neither rCPBm4 nor rSUMO-CPBm4 was lethal to mice. Although rCPBm4 and rSUMO-CPBm4 were reactogenic with polyclonal antibodies against crude CPB, rabbits vaccinated with rSUMO-CPBm4 developed significant levels of toxin-neutralizing antibody (TNA) titers that conferred protection against crude toxin challenge. These data suggest that genetically detoxified rSUMO-CPBm4 is a promising subunit vaccine candidate for C. perfringens type C beta enterotoxemia.
    Matched MeSH terms: Recombinant Proteins/genetics
  6. 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
  7. Ramachandram DS, Kow CS, Hasan SS
    J Cyst Fibros, 2023 May;22(3):580.
    PMID: 36966036 DOI: 10.1016/j.jcf.2023.03.010
    Matched MeSH terms: Recombinant Proteins/therapeutic use
  8. Wong RS, Alias NNM, Ong EBB, Liew MWO
    Methods Mol Biol, 2023;2617:189-200.
    PMID: 36656525 DOI: 10.1007/978-1-0716-2930-7_13
    Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.
    Matched MeSH terms: Recombinant Proteins/chemistry
  9. Abdo AIK, Ngoh YY, Lew MH, Dass SA, Rahumatullah A, Noordin R, et al.
    Biotechnol Appl Biochem, 2022 Feb;69(1):70-76.
    PMID: 33258152 DOI: 10.1002/bab.2082
    Lymphatic filariasis is a neglected parasitic disease that affects millions in tropical and subtropical countries and is caused by Wuchereria and Brugia species. Specific and sensitive detection methods are essential in mapping infected areas where rapid tests are needed to cover underdeveloped and remote regions, which facilitates eliminating the disease as a public health problem. A few commercialized rapid tests based on antigen or antibody detection are available, but the former only detects infection by Wuchereria species and cross-reacts with nonlymphatic filaria, whereas antibody detection might provide positive results of previous infection. Here, we report the production of three different recombinant immunoglobulin gamma (IgG)1 antibodies based on scFvs previously generated via human antibody phage display technology, that is, anti-BmR1 clone 4, anti-BmXSP clone 5B, and anti-BmXSP clone 2H2. The scFv sequences were cloned into a pCMV-IgG1 vector, then transfected into a HEK293F cell line. The generated antibodies were found to be able to bind to their respective targets even at relatively low concentration. Conjugation of Fc to scFv induces binder stability and provides multiple labeling sites for probes and signaling molecules that can be used in rapid tests.
    Matched MeSH terms: Recombinant Proteins
  10. Azemi NFH, Misnan R, Keong BP, Mokhtar M, Kamaruddin N, Fah WC, et al.
    Mol Biol Rep, 2021 Oct;48(10):6709-6718.
    PMID: 34427887 DOI: 10.1007/s11033-021-06661-x
    BACKGROUND: Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity.

    METHODS AND RESULTS: The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients.

    CONCLUSIONS: This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

    Matched MeSH terms: Recombinant Proteins/genetics*; Recombinant Proteins/immunology*
  11. Chong WL, Chupradit K, Chin SP, Khoo MM, Khor SM, Tayapiwatana C, et al.
    Molecules, 2021 Sep 20;26(18).
    PMID: 34577167 DOI: 10.3390/molecules26185696
    Protein-protein interaction plays an essential role in almost all cellular processes and biological functions. Coupling molecular dynamics (MD) simulations and nanoparticle tracking analysis (NTA) assay offered a simple, rapid, and direct approach in monitoring the protein-protein binding process and predicting the binding affinity. Our case study of designed ankyrin repeats proteins (DARPins)-AnkGAG1D4 and the single point mutated AnkGAG1D4-Y56A for HIV-1 capsid protein (CA) were investigated. As reported, AnkGAG1D4 bound with CA for inhibitory activity; however, it lost its inhibitory strength when tyrosine at residue 56 AnkGAG1D4, the most key residue was replaced by alanine (AnkGAG1D4-Y56A). Through NTA, the binding of DARPins and CA was measured by monitoring the increment of the hydrodynamic radius of the AnkGAG1D4-gold conjugated nanoparticles (AnkGAG1D4-GNP) and AnkGAG1D4-Y56A-GNP upon interaction with CA in buffer solution. The size of the AnkGAG1D4-GNP increased when it interacted with CA but not AnkGAG1D4-Y56A-GNP. In addition, a much higher binding free energy (∆GB) of AnkGAG1D4-Y56A (-31 kcal/mol) obtained from MD further suggested affinity for CA completely reduced compared to AnkGAG1D4 (-60 kcal/mol). The possible mechanism of the protein-protein binding was explored in detail by decomposing the binding free energy for crucial residues identification and hydrogen bond analysis.
    Matched MeSH terms: Recombinant Proteins/metabolism*; Recombinant Proteins/chemistry
  12. Noordin R, Anuar NS, Juri NM, Wongphutorn P, Ruantip S, Kopolrat KY, et al.
    Am J Trop Med Hyg, 2021 07 08;105(3):688-691.
    PMID: 34237022 DOI: 10.4269/ajtmh.21-0317
    Strongyloides stercoralis affects more than half a billion people worldwide, and hyperinfection in immunocompromised patients can be fatal. Elimination of this neglected tropical disease requires field-applicable diagnostic tools. We conducted a laboratory evaluation of a lateral flow rapid dipstick test (SsRapid™) using sera samples from a Strongyloides-endemic area in northeast Thailand. Group 1 was S. stercoralis-positive and larvae- and/or antibody-positive (according to the IgG ELISA) (N = 100). Group 2 had negative fecal examination and IgG ELISA results (N = 25). Group 3 had other parasitic infections and negative IgG ELISA results (N = 25). The results showed good diagnostic sensitivity (82%) and excellent specificity (96%). Suggested improvements in the SsRapid™ test include increased diagnostic sensitivity and conversion to the more robust cassette format. Field studies should be performed as well.
    Matched MeSH terms: Recombinant Proteins
  13. Muhammed NS, Hussin N, Lim AS, Jonet MA, Mohamad SE, Jamaluddin H
    Protein J, 2021 06;40(3):419-435.
    PMID: 33870461 DOI: 10.1007/s10930-021-09986-5
    Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 β-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/β motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/chemistry
  14. Ejike UC, Chan CJ, Lim CSY, Lim RLH
    Appl Microbiol Biotechnol, 2021 Apr;105(7):2799-2813.
    PMID: 33763709 DOI: 10.1007/s00253-021-11225-x
    Fungal immunomodulatory proteins (FIPs) are bioactive proteins with immunomodulatory properties. We previously reported the heterologous production in Escherichia coli of FIP-Lrh from Tiger milk mushroom (Lignosus rhinocerus) with potent cytotoxic effect on cancer cell lines. However, protein produced in E. coli lacks post-translational modifications and may be contaminated with lipopolysaccharide (LPS) endotoxin. Therefore, in this study, yFIP-Lrh produced in Pichia pastoris was functionally compared with eFIP-Lrh produced in E. coli. Expression construct of FIP-Lrh cDNA in pPICZα was generated, transformed into P. pastoris X-33 and Mut+ transformants were verified by colony PCR. Induction with 0.5% or 1% methanol resulted in a secreted 13.6 kDa yFIP-Lrh which was subsequently purified and verified using LCMS/MS analysis. Size exclusion chromatography confirmed eFIP-Lrh as a homodimer whereas the larger size of yFIP-Lrh may indicate post-translational modification despite negative for glycoproteins staining. At lower concentration (4-8 μg/mL), yFIP-Lrh induced significantly higher Th1 (IFN-γ, TNF-α) and Th2 (IL-6, IL-4, IL-5, IL-13) cytokines production in mice splenocytes, whereas 16 μg/mL eFIP-Lrh induced significantly higher pro-inflammatory cytokines (TNF-α, IL-6, IL-10), possibly due to higher residual LPS endotoxin (0.082 EU/mL) in eFIP-Lrh compared to negligible level in yFIP-Lrh (0.001 EU/mL). Furthermore, yFIP-Lrh showed higher cytotoxic effect on MCF-7 and HeLa cancer cells. Since both recombinant proteins of FIP-Lrh have the same peptide sequence, besides glycosylation, other post-translational modifications in yFIP-Lrh may account for its enhanced immunomodulatory and anti-proliferative activities. In conclusion, P. pastoris is preferred over E. coli for production of a functionally active yFIP-Lrh devoid of endotoxin contamination. KEY POINTS: • FIP-Lrh can induced production of Th1 and Th2 cytokines by mouse splenocytes. • Higher cytotoxic effect on cancer cells observed for yeast compared to E. coli produced FIP-Lrh. • P. pastoris allows production of an endotoxin-free and functionally active recombinant FIP-Lrh.
    Matched MeSH terms: Recombinant Proteins/genetics
  15. Ng BH, Mohd Aminudin NH, Nasaruddin MZ, Abdul Rahaman JA
    BMJ Case Rep, 2021 Feb 05;14(2).
    PMID: 33547099 DOI: 10.1136/bcr-2020-239702
    Patients with symptomatic complex malignant pleural effusion (MPE) are frequently unfit for decortication and have a poorer prognosis. Septations can develop in MPE, which may lead to failure of complete drainage and pleural infection. Intrapleural fibrinolytic therapy (IPFT) is an alternative treatment. The use of IPFT in patients with anaemia and high risk for intrapleural bleeding is not well established. We report a successful drainage of complex haemoserous MPE with a single modified low-dose of intrapleural 5 mg of alteplase and 5 mg of dornase alfa in a patient with pre-existing anaemia with no significant risk of intrapleural bleeding.
    Matched MeSH terms: Recombinant Proteins/therapeutic use
  16. Mohd Ali MR, Sum JS, Aminuddin Baki NN, Choong YS, Nor Amdan NA, Amran F, et al.
    Int J Biol Macromol, 2021 Jan 31;168:289-300.
    PMID: 33310091 DOI: 10.1016/j.ijbiomac.2020.12.062
    Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.
    Matched MeSH terms: Recombinant Proteins/genetics
  17. Rahumatullah A, Balachandra D, Noordin R, Baharudeen Z, Lim YY, Choong YS, et al.
    Sci Rep, 2021 01 28;11(1):2502.
    PMID: 33510342 DOI: 10.1038/s41598-021-82125-3
    Antibodies have different chemical properties capable of targeting a diverse nature of antigens. Traditionally, immune antibody libraries are perceived to be disease-specific with a skewed repertoire. The complexity during the generation of a combinatorial antibody library allows for a skewed but diverse repertoire to be generated. Strongyloides stercoralis is a parasite that causes strongyloidiasis, a potentially life-threatening disease with a complex diagnosis that impedes effective control and treatment of the disease. This study describes the isolation of monoclonal antibodies against S. stercoralis NIE recombinant protein using an immune antibody phage display library derived from lymphatic filaria-infected individuals. The isolated antibody clones showed both lambda and kappa light chains gene usage, with diverse amino acid distributions. Structural analysis showed that electropositivity and the interface area could determine the binding affinity of the clones with NIE. The successful identification of S. stercoralis antibodies from the filarial immune library highlights the breadth of antibody gene diversification in an immune antibody library that can be applied for closely related infections.
    Matched MeSH terms: Recombinant Proteins/genetics
  18. Tee YN, Kumar PV, Maki MAA, Elumalai M, Rahman SAKMEH, Cheah SC
    Curr Pharm Biotechnol, 2021;22(7):969-982.
    PMID: 33342408 DOI: 10.2174/1389201021666201218124450
    BACKGROUND: Recombinant Keratinocyte Growth Factor (rHuKGF) is a therapeutic protein used widely in oral mucositis after chemotherapy in various cancers, stimulating lung morphogenesis and gastrointestinal tract cell proliferation. In this research study, chitosan-rHuKGF polymeric complex was implemented to improve the stability of rHuKGF and used as rejuvenation therapy for the treatment of oral mucositis in cancer patients.

    OBJECTIVE: Complexation of rHuKGF with mucoadhesive low molecular weight chitosan to protect rHuKGF from proteolysis and investigate the effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells.

    METHODS: The interaction between chitosan and rHuKGF was studied by molecular docking. Malvern ZetaSizer Nano Zs and Fourier-Transform Infrared spectroscopy (FTIR) tests were carried out to characterize the chitosan-rHuKGF complex. In addition, SDS-PAGE was performed to investigate the interaction between chitosan-rHuKGF complex and pepsin. The effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells was studied by MTT assay.

    RESULTS: Chitosan-rHuKGF complex was formed through the hydrogen bonding proven by the docking studies. A stable chitosan-rHuKGF complex was formed at pH 4.5 and was protected from proteolysis and assessed by SDS PAGE. According to the MTT assay results, chitosan-rHuKGF complex increased the cell proliferation rate of FHs 74 Int cells.

    CONCLUSION: The developed complex improved the stability and the biological function of rHuKGF.

    Matched MeSH terms: Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  19. Abu ML, Mohammad R, Oslan SN, Salleh AB
    Prep Biochem Biotechnol, 2021;51(4):350-360.
    PMID: 32940138 DOI: 10.1080/10826068.2020.1818256
    A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*
  20. Jambari NN, Liddell S, Martinez-Pomares L, Alcocer MJC
    PLoS One, 2021;16(4):e0249876.
    PMID: 33914740 DOI: 10.1371/journal.pone.0249876
    Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/immunology; Recombinant Proteins/isolation & purification
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