Biotechnologia最新文献

{"title":"Expression of the gene encoding blood coagulation factor VIII without domain B in <i>E. coli</i> bacterial expression system.","authors":"Anna Mazurkiewicz-Pisarek,&nbsp;Alina Mazurkiewicz,&nbsp;Diana Mikiewicz,&nbsp;Piotr Baran,&nbsp;Tomasz Ciach","doi":"10.5114/bta.2023.130728","DOIUrl":"10.5114/bta.2023.130728","url":null,"abstract":"In this article, we have demonstrated the feasibility of generating an active form of recombinant blood coagulation factor VIII using an E. coli bacterial expression system as a potential treatment for hemophilia type A. Factor VIII (FVIII), an essential blood coagulation protein, is a key component of the fluid phase blood coagulation system. So far, all available recombinant FVIII formulations have been produced using eukaryotic expression systems. Mammalian cells can produce catalytically active proteins with all the necessary posttranslational modifications. However, cultivating such cells is time-consuming and highly expensive, and the amount of the obtained product is usually low. In contrast to eukaryotic cells, bacterial culture is inexpensive and allows the acquisition of large quantities of recombinant proteins in a short time. With this study, we aimed to obtain recombinant blood coagulation factor VIII using the E. coli bacterial expression system, a method not previously explored for this purpose. Our research encompasses the synthesis of blood coagulation factor VIII and its expression in a prokaryotic system. To achieve this, we constructed a prokaryotic expression vector containing a synthetic factor VIII gene, which was then used for the transformation of an E. coli bacterial strain. The protein expression was confirmed by mass spectrometry, and we assessed the stability of the gene construct while determining the optimal growth conditions. The production of blood coagulation factor VIII by the E. coli bacterial strain was carried out on a quarter-technical scale. We established the conditions for isolation, denaturation, and renaturation of the protein, and subsequently confirmed the activity of FVIII.","PeriodicalId":94371,"journal":{"name":"Biotechnologia","volume":"104 3","pages":"247-262"},"PeriodicalIF":0.0,"publicationDate":"2023-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c5/b5/BTA-104-3-51301.PMC10578111.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41242919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effect of sucrose as an elicitor in increasing quercetin-3-O-rhamnoside (quercitrin) content of chrysanthemum (<i>Chrysanthemum morifolium</i> Ramat) callus culture based on harvest time differences.","authors":"Tia Setiawati,&nbsp;Annisa N Arofah,&nbsp;Mohamad Nurzaman,&nbsp;Annisa Annisa,&nbsp;Asep Z Mutaqin,&nbsp;Rusdi Hasan","doi":"10.5114/bta.2023.130731","DOIUrl":"10.5114/bta.2023.130731","url":null,"abstract":"<p><p>Chrysanthemum (<i>Chrysanthemum morifolium</i>) contains secondary metabolites, such as flavonoid compounds, especially luteolin-7-glucoside and quercetin-3-O-rhamnoside (quercitrin), in its tissues. Utilizing sucrose as an elicitor through callus culture presents an alternative method to enhance the production of secondary metabolites. This research aimed to determine the best sucrose concentration and harvest time for maximizing quercitrin content in chrysanthemum callus culture. The research employed a completely randomized design with four treatment groups: 0, 30, 45, and 60 g/l of sucrose added to MS medium containing 4 ppm 2,4-dichlorophenoxyacetic acid (2,4-D). Callus samples were harvested on the 15th and 30th days of culture. The observed parameters included callus morphology (color and texture), fresh weight, dry weight, the diameter of the callus, and quercitrin content analyzed using high-performance liquid chromatography. The results showed that all callus cultures exhibited intermediate textures and varied colors, predominantly shades of brown. The treatment involving 45 g/l of sucrose with a 30th-day harvest yielded the highest fresh weight, dry weight, and quercitrin content, namely 2.108 g, 0.051 g, and 0.437 mg/g DW, respectively. Notably, the quercitrin content exhibited a 63.67% increase compared to the control.</p>","PeriodicalId":94371,"journal":{"name":"Biotechnologia","volume":"104 3","pages":"289-300"},"PeriodicalIF":0.0,"publicationDate":"2023-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d3/53/BTA-104-3-51304.PMC10578125.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41242918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of potential CD8+ epitopes in pp62 polyprotein of African swine fever virus using computational immunology.","authors":"Mark Lester C Galicia,&nbsp;Dale Jonathan M Morales,&nbsp;Precious Grace B Pogado,&nbsp;Ashley L Quebrado,&nbsp;Leana Rich Herrera-Ong","doi":"10.5114/bta.2023.130726","DOIUrl":"10.5114/bta.2023.130726","url":null,"abstract":"<p><p>The highly infectious African swine fever virus (ASFV) is currently the only known DNA arbovirus within the <i>Asfarviridae</i> family which primarily infects domestic pigs and wild boars. African swine fever (ASF) leads to a mortality rate of up to 100% which has caused massive socio-economic losses worldwide. Previous research indicates that ASFV's virulence can be attributed to polyprotein pp62, which plays a crucial role in viral assembly and core maturation. This particular study utilized <i>in silico</i> analysis to identify highly conserved cytotoxic T-cell epitopes in pp62 that can potentially serve as key components for future ASFV vaccines. To achieve this, the researchers retrieved, clustered, and aligned the peptide sequences of pp62. Subsequently, the aligned sequences were analyzed to identify epitopes that bind promiscuously to the swine major histocompatibility complex I (MHC I) alleles and exhibiting MHC IC₅₀ values < 500 nM. Additionally, peptide sequences with positive proteasome and TAP scores were considered. Potential cross-reactivity was assessed by comparing the peptide sequences against available proteome sequences of <i>Sus scrofa domesticus</i> in various databases. Furthermore, molecular docking was conducted to evaluate the binding of candidate epitopes with swine leukocyte antigen-1*0401 (SLA-1*0401). The dissociation constants, binding energies, root mean square deviation, and root mean square fluctuation values for the SLA-epitope complexes were compared with a positive reference. In the course of the study, 21 highly conserved CD8+ epitopes were identified, out of which four were further assessed for their potential immunogenicity. The results demonstrated that the highly conserved CD8+ epitopes discovered in this study are promising for integration into future ASFV vaccine formulations. As preliminary data, it is anticipated that these findings will subsequently undergo <i>in vitro</i> and <i>in vivo</i> studies in the future.</p>","PeriodicalId":94371,"journal":{"name":"Biotechnologia","volume":"104 3","pages":"221-231"},"PeriodicalIF":0.0,"publicationDate":"2023-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/92/c8/BTA-104-3-51299.PMC10578124.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41242921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}