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To battle seasonal outbreaks of influenza B virus infection, which continue to pose a major threat to world health, new and improved vaccines are urgently needed. In this article, we discuss the current state of next-generation influenza B vaccine development, including both advancements and challenges. This review covers the shortcomings of existing influenza vaccines and stresses the need for more-effective and broadly protective vaccines and more-easily scalable manufacturing processes. New possibilities for vaccine development have emerged due to recent technical developments such as virus-like particle (VLP) platforms, recombinant DNA technologies, and reverse genetics. By using these methods, vaccines can be developed that elicit stronger and longer-lasting immune responses against various strains of influenza B virus. Vaccines may be more effective and immunogenic when adjuvants and new delivery mechanisms are used. Progress has been made in the development of influenza B vaccine mRNA vaccines, nanoparticle-based vaccines, and vector-based vaccines. However, there are still several obstacles to overcome before next-generation influenza B vaccines can be widely used, including the challenge of antigenic drift, the extinction of the B/Yamagata lineage, and difficulties in strain selection. There are also other challenges related to public acceptance, vaccine distribution, manufacturing complexity, and regulations. To overcome these challenges, scientists, politicians, and pharmaceutical firms must work together to expedite the development and licensing of vaccines and the establishment of immunization programs. The need for constant monitoring and quick adaptation of vaccines to match the currently circulating strains is further highlighted by the appearance of novel influenza B virus variants. To be ready for future pandemics and influenza B outbreaks, we need better vaccines and better monitoring systems. [ABSTRACT FROM AUTHOR]

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of the last part of this review article. [ABSTRACT FROM AUTHOR]

This study determined the effects of administering a glycoprotein with equine chorionic gonadotropin (eCG)-like activity (eCG-like) on corpus luteum (CL) area, serum progesterone concentrations, incidence of multiple ovulations (MOV), estrus expression rate (EER), and pregnancy to timed AI (P/TAI) in Angus cattle synchronized with a 5-d Co-Synch protocol. On Day −8, cattle were body condition scored (BCS), and received a 1.0 g progesterone intravaginal device (IVD) and 100 μg GnRH. On Day −3, the IVDs were removed and 500 μg cloprostenol was administered intramuscularly (i.m.). Cattle were randomly assigned into one of two groups: eCG-like (heifers, n = 232, primiparous, n = 148, and multiparous cows = 485; 300 IU (heifers) and 400 IU (cows) eCG-like i.m. on Day −3), or Control (heifers, n = 240, primiparous, n = 151, and multiparous cows, n = 478; no eCG-like). On Day −2, cattle received a second dose of 500 μg cloprostenol, and on Day 0, 100 μg GnRH was given concurrently with TAI. Estrus expression rate was assessed by observing the tail paint rubbed off in a subset of heifers (n = 372) and all cows on Day 0. Transrectal ultrasonography was used to evaluate the presence of CL on Day −8 and to diagnose P/TAI on Day 30–35. In a subset of cattle (heifers = 194 and multiparous cows = 87), CL area, serum progesterone concentrations, and incidence of MOV were evaluated on Day 7. Heifers, primiparous, and multiparous cows were analyzed separately. Treatment with eCG-like did not affect (P > 0.1) EER in heifers. Estrus expression rate was increased (P ≤ 0.03) in primiparous (68.9 % vs 45.0 %) and multiparous (75.5 % vs. 68.8 %) cows treated with eCG-like compared with Controls. Pregnancy/TAI was increased (P < 0.01) in heifers (65.2 % vs 48.3 %) and primiparous cows (48.3 % vs. 35.1 %) treated with eCG-like than Controls. In multiparous cows with a BCS ≤4 P/TAI was increased (P = 0.03) in the eCG-like group (47.7 %) than the Control group (34.8 %) but was similar (P > 0.1) between treatment groups in multiparous cows with a BCS ≥4.5. The eCG-like treatment increased (P < 0.05) CL area in heifers and multiparous cows and tended (P = 0.10) to elevate serum progesterone concentrations only in heifers. However, it did not affect (P > 0.1) the incidence of MOV in heifers and multiparous cows. Glycoprotein eCG-like administration increased fertility in heifers and primiparous cows, but in multiparous the effect of eCG-like on fertility was associated with BCS. eCG-like treatment in a 5-day Co-Synch synchronization protocol: • Increases estrus expression rate in primiparous and multiparous cows. • Increases P/TAI in heifers, primiparous cows, and multiparous cows with a BCS ≤ 4. • Increases CL area in heifers and multiparous cows. • Does not affect the incidence of multiple ovulations in heifers and multiparous cows. [ABSTRACT FROM AUTHOR]

Escherichia coli has shouldered a massive workload with the discovery of recombinant DNA technology. A new era began in the biopharmaceutical industry with the production of insulin, the first recombinant protein, in E. coli and its use in treating diabetes. After insulin, many biopharmaceuticals produced from E. coli have been approved by the US Food and Drug Administration and the European Medicines Agency to treat various human diseases. Although E. coli has some disadvantages, such as lack of post‐translational modifications and toxicity, it is an important host with advantages such as being a well‐known bacterium in recombinant protein production, cheap, simple production system, and high yield. This study examined biopharmaceuticals produced and approved in E. coli under the headings of peptides, hormones, enzymes, fusion proteins, antibody fragments, vaccines, and other pharmaceuticals. The topics on which these biopharmaceuticals were approved for treating human diseases, when and by which company they were produced, and their use and development in the field are included. [ABSTRACT FROM AUTHOR]

Lipids are primarily transported in the bloodstream by lipoproteins, which are macromolecules of lipids and conjugated proteins also known as apolipoproteins. The processes of lipoprotein assembly, secretion, transportation, modification, and clearance are crucial components of maintaining a healthy lipid metabolism. Disruption in any of these steps results in pathophysiological abnormalities such as dyslipidemia, obesity, insulin resistance, inflammation, atherosclerosis, peripheral artery disease, and cardiovascular diseases. By studying these genetic mutations, researchers can gain valuable insights into the underlying mechanisms that govern the relationship between protein structure and its physiological role. These lipoproteins, including HDL, LDL, lipoprotein(a), and VLDL, mainly serve the purpose of transporting lipids between tissues and organs. However, studies have provided evidence that apo(a) also possesses protective properties against pathogens. In the future, the field of study will be significantly influenced by the integration of recombinant DNA technology and human site-specific mutagenesis for treating hereditary disorders. Several medications are available for the treatment of dyslipoproteinemia. These include statins, fibrates, ezetimibe, niacin, PCSK9 inhibitors, evinacumab, DPP 4 inhibitors, glucagon-like peptide-1 receptor agonists GLP1RAs, GLP-1, and GIP dual receptor agonists, in addition to SGLT2 inhibitors. This current review article exhibits, for the first time, a comprehensive reflection of the available body of publications concerning the impact of lipoproteins on metabolic well-being across various pathological states. [ABSTRACT FROM AUTHOR]

In the late 1970s, crude interferon samples were found to exhibit anti-tumour activity. This discovery led to the interferon as a “magic drug” for cancer patients. Many groups, including those in Tokyo, Zurich, and San Francisco, attempted to identify human interferon cDNAs. Tadatsugu Taniguchi was the first to announce the cloning of human interferon-β cDNA in the December 1979 issue of Proc. Jpn. Acad. Ser. B. This was followed by the cloning of human interferon-α by a Zurich group and interferon-γ by a group in Genentech in San Francisco. Recombinant interferon proteins were produced on a large scale, and interferon-α was widely used to treat C-type hepatitis patients. The biological functions of interferons were quickly elucidated with the purified recombinant interferons. The molecular mechanisms underlying virus-induced interferon gene expression were also examined using cloned chromosomal genes. The background that led to interferon gene cloning and its impact on cytokine gene hunting is described herein.

The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity. [ABSTRACT FROM AUTHOR]

Streptokinase (SK) is one of the essential fibrinolytic agents and is commonly employed to treat myocardial infarction or heart attack. Despite its significance as an anti-clot agent, SK exhibits certain drawbacks, including limited half-life in the bloodstream, non-specific to fibrin, and may cause process workers to develop undesirable immune responses. Acknowledging the high specificity of staphylokinase (SAK) towards fibrin and the powerful recombinant DNA technology, this study was carried out to evaluate the fibrinolytic activity of a recombinant staphylokinase-streptokinase (SAK-SK) protein in Escherichia coli BL21(DE3). Briefly, the SAK gene isolated from Staphylococcus aureus was ligated at the 5'end of the SK gene using the pGEM®-3Zf (+) cloning vector and transformed into the expression host E. coliBL21(DE3) before being induced with isopropyl β-D-1-thiogalactopyranoside (IPTG).Subsequently, the produced recombinant SK protein was analyzed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The biological performance of the recombinant SK protein was then examined via caseinolytic and fibrinolytic assays and compared to that of the native SK protein. Based on the results, the recombinant SK protein contained 563 amino acid residues with a molecular weight of approximately 63.1 kDa. The hybrid SK protein was more active in the casein lysis experiment compared to the native SK, with a more significant halo zone formation. Moreover, the hybrid SK protein demonstrated a complete clot lysis compared to the poor clot lysis of the native SK protein. In conclusion, this study successfully developed a recombinant hybrid SK protein with enhanced affinity to fibrin, making it a potential anti-clot agent. [ABSTRACT FROM AUTHOR]

In the last two decades, protein–protein interactions (PPIs) have been used as the main target for drug development. However, with larger or superficial binding sites, it has been extremely difficult to disrupt PPIs with small molecules. On the other hand, intracellular PPIs cannot be targeted by antibodies that cannot penetrate the cell membrane. Peptides that have a combination of conformational rigidity and flexibility can be used to target difficult binding interfaces with appropriate binding affinity and specificity. Since the introduction of insulin nearly a century ago, more than 80 peptide drugs have been approved to treat a variety of diseases. These include deadly diseases such as cancer and human immunodeficiency virus infection. It is also useful against diabetes, chronic pain, and osteoporosis. Today, more research is being done on these drugs as lessons learned from earlier approaches, which are still valid today, complement newer approaches such as peptide display libraries. At the same time, integrated genomics and peptide display libraries are new strategies that open new avenues for peptide drug discovery. The purpose of this review is to examine the problems in elucidating the peptide‐protein recognition mechanism. This is important to develop peptide‐based interventions that interfere with endogenous protein interactions. New approaches are being developed to improve the binding affinity and specificity of existing approaches and to develop peptide agents as potentially useful drugs. We also highlight the key challenges that must be overcome in peptide drug development to realize their potential and provide an overview of recent trends in peptide drug development. In addition, we take an in‐depth look at early efforts in human hormone discovery, smart medicinal chemistry and design, natural peptide drugs, and breakthrough advances in molecular biology and peptide chemistry. [ABSTRACT FROM AUTHOR]

Many universities resort to online teaching due to the COVID-19 pandemic. It is a challenging endeavour, especially in Biology courses that require lab access. Mock grant application roleplay is one alternative to lab-based activities. Although using mock grant applications as an assessment tool is not new, there have been few studies on students’ opinions. To the best of our knowledge, this is the first time that it has been used in place of lab-based exercises and in conjunction with virtual lab modules. Students are engaged in three aspects: (i) targeted literature review, (ii) research proposal writing and (iii) 5-min project pitching. The design of this module is flexible, and other lab-based courses can adopt it. This module encourages undergraduate students to explore the lab techniques and concisely present their research proposals. Compared to the previous semester before COVID-19, the number of students that achieved the “Distinction” grade or higher increased by 6.3%, whilst the failures decreased by 3.2%. A similar trend was observed in 2021, the second year this activity was carried out. A survey amongst students who took this unit reported that student satisfaction with this unit has improved by 11.1%. This improvement could be attributed to this mock grant activity because the format and difficulty level of the student assessments had remained constant. Furthermore, qualitative analysis conducted via focus group interviews indicated that students agreed that the mock grant proposal assessment was useful in preparing them for future careers and was relevant to the course learning outcomes. Several participants pointed to the assessment’s potential usefulness for careers in research. In conclusion, this roleplay module can fulfil the learning objectives of this course whilst providing an authentic research experience without lab-based activities.

Along with the wide applications of conventional plastics, they have a large number of disadvantages like their non-biodegradable nature, dependency on fossil fuels and the release of large amounts of toxic materials in the environment. Therefore, to resolve these problems, a number of bioplastics are studied, out of which polyhydroxyalkanoates are considered as the best alternatives. Polyhydroxyalkanoates (PHAs) are produced by microorganisms as intracellular granules during stressful conditions. Though a wide range of organisms can naturally produce PHAs, only a few of them can be used for commercial production. Therefore, more diverse organisms that accumulate a considerable amount of PHAs and also reduce the production cost need to be exploited. Transgenic plants, recombinant bacteria, algae and extremophiles are some diverse organisms that produce a high amount of PHAs at a low cost. So, if potential organisms are used for PHA production, bioplastics will be able to completely replace petroleum-based polymers. Therefore, our review mainly focuses on production of PHAs using potential organisms so that amount of PHAs produced is high and cost-effective which would further help in the commercialization of PHAs. Highlights: Properties like biodegradability, environmental protection and sustainable nature make polyhydroxyalkanoates a good commercial alternative to non-biodegradable plastics. Production of PHA using diversity of organisms like extremophiles, recombinant bacteria, algae and transgenic plants will help in producing PHAs at a lower cost with a much higher yield. By using microorganisms which are capable of producing high amount of PHA, it is expected that PHAs will be able to compete with petroleum-based polymers commercially and will be capable of completely replacing them. [ABSTRACT FROM AUTHOR]

Viral infectious diseases threaten human health and global stability. Several vaccine platforms, such as DNA, mRNA, recombinant viral vectors, and virus-like particle-based vaccines have been developed to counter these viral infectious diseases. Virus-like particles (VLP) are considered real, present, licensed and successful vaccines against prevalent and emergent diseases due to their non-infectious nature, structural similarity with viruses, and high immunogenicity. However, only a few VLP-based vaccines have been commercialized, and the others are either in the clinical or preclinical phases. Notably, despite success in the preclinical phase, many vaccines are still struggling with small-scale fundamental research owing to technical difficulties. Successful production of VLP-based vaccines on a commercial scale requires a suitable platform and culture mode for large-scale production, optimization of transduction-related parameters, upstream and downstream processing, and monitoring of product quality at each step. In this review article, we focus on the advantages and disadvantages of various VLP-producing platforms, recent advances and technical challenges in VLP production, and the current status of VLP-based vaccine candidates at commercial, preclinical, and clinical levels.

Influenza is a vaccine-preventable disease. Due to the evolving nature of influenza viruses, the composition of vaccines has to be updated annually. Most of the current influenza vaccines are still produced in embryonated chicken eggs, a well-established process with some limitations. This review focuses on the recombinant DNA technology using baculovirus expression vector system a modern method of manufacturing licensed influenza vaccines. The speed, scalability, biosafety and flexibility of the process, together with the reliability of the hemagglutinin in the vaccine, represent a significant advance toward new platforms for vaccine production. The scenario of vaccine production in the next years seems to be particularly interesting, involving a transition from the current egg-based production to new technologies, such as the cell culture platform, the RNA technology, the plant-based system, and the DNA vaccine. This latter offers great advantages over egg- and cell-based influenza vaccine production. The universal vaccine remains the goal of researchers and ideally would avoid the need for annual reformulation and re-administration of seasonal vaccines. The lesson learned from the COVID-19 pandemic highlights the importance of having different technologies available and able to promptly respond to a great demand of vaccines worldwide. [ABSTRACT FROM AUTHOR]

Proteases are closely associated with many pathological conditions. Efficient detection of protease activity may be useful for diagnosis, prognosis, and the development of new therapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as the non-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins are widely used in FRET biosensors because they can be genetically encoded and compatible with cells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. In this study, it was aimed to detect protease activity using an FP-FRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding the mNeonGreen-mRuby3 fluorescent protein-based FRET biosensor was constructed. The gene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells using recombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed in the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.

Biologics are the molecules of protein, produced by recombinant DNA technology that are used in various diseases to target the specific points in the immunopathogenesis of the diseases without interfering with rest of the pathogenetic pathways and thereby expected to have a lesser side effects profile when compared to conventional immunosuppressant. With the advent of biologic therapy, the treatment of various systemic and cutaneous diseases, especially autoimmune diseases, has been revolutionized; biologicals are goal-directed lethal weapons in treatment armamentarium of a dermatologist; they are a major breakthrough and game changer in the field of dermatology therapeutics or you can say in general as a therapeutic option. While adults have been enjoying the benefits of biologics in treating skin conditions such as psoriasis, eczema, and hidradenitis suppurativa for last few years, but the use of biologics in the pediatric population is still limited because of unknown long-term safety profile and absence of large-scale studies. Thankfully, times are changing, and Biologics are slowly being approved for pediatric use too, in coming future dermatologists will be able to assess which pathways, in particular, are overactive and then prescribing the exact biological as per the need of the patient. In this review, a brief description is given of different biologic agents that are known currently. An extensive literature search was done; all clinical trials, randomized double-blind or single-blind controlled trials, open-label studies, retrospective studies, reviews, case series, and case reports concerned with the use of biologics in various pediatrics dermatoses were screened. The selected articles were retrieved; the final manuscript was prepared, analyzed, and presented in a narrative fashion.

Worldwide, environmental pollution due to a complex mixture of xenobiotics has become a serious concern. Several xenobiotic compounds cause environmental contamination due to their severe toxicity, prolonged exposure, and limited biodegradability. From the past few decades, microbial-assisted degradation (bioremediation) of xenobiotic pollutants has evolved as the most effective, eco-friendly, and valuable approach. Microorganisms have unique metabolism, the capability of genetic modification, diversity of enzymes, and various degradation pathways necessary for the bioremediation process. Microbial xenobiotic degradation is effective but a slow process that limits its application in bioremediation. However, the study of microbial enzymes for bioremediation is gaining global importance. Microbial enzymes have a huge ability to transform contaminants into non-toxic forms and thereby reduce environmental pollution. Recently, various advanced techniques, including metagenomics, proteomics, transcriptomics, metabolomics are effectively utilized for the characterization, metabolic machinery, new proteins, metabolic genes of microorganisms involved in the degradation process. These advanced molecular techniques provide a thorough understanding of the structural and functional aspects of complex microorganisms. This review gives a brief note on xenobiotics and their impact on the environment. Particular attention will be devoted to the class of pollutants and the enzymes such as cytochrome P450, dehydrogenase, laccase, hydrolase, protease, lipase, etc. capable of converting these pollutants into innocuous products. This review attempts to deliver knowledge on the role of various enzymes in the biodegradation of xenobiotic pollutants, along with the use of advanced technologies like recombinant DNA technology and Omics approaches to make the process more robust and effective. [ABSTRACT FROM AUTHOR]

The paper provides an overview of concepts and knowledge about genetic engineering and its development in Croatia and abroad from the second half of the 20th century to the present day. Genetic engineering is the biological term that has undergone the greatest conceptual deformations, and therefore some important concepts are unambiguously explained in the paper. As data and documentation on this are not widely available to the public, a systematic overview of the development of genetic engineering in Croatia as well as the roles of individuals, their interactions and contributions is also presented. As early as the 1970s, the Croatian pharmaceutical industry and research institutions in Croatia produced a wave of biotechnological and genetic engineering research and product development. Various research groups, mostly gathered around the constituents of the University of Zagreb (especially the Faculty of Food Technology and Biotechnology and the Faculty of Science), the Ruđer Bošković Institute and PLIVA, use recombinant DNA technology for their basic research. In addition to a chronology of events related to genetic engineering, the paper also describes the work and merits of scientists M. Demerec, V. Johanides, M. Alačević, Ž. Trgovčević, Ž. Kućan, S. Jelaska, V. Gamulin, V. Delić, I. Weygand-Đurašević, Z. Zgaga, S. Jelenić, and others in the field of genetic engineering.

The first leptospiral recombinant vaccine was developed in the late 1990s. Since then, progress in the fields of reverse vaccinology (RV) and structural vaccinology (SV) has significantly improved the identification of novel surface-exposed and conserved vaccine targets. However, developing recombinant vaccines for leptospirosis faces various challenges, including selecting the ideal expression platform or delivery system, assessing immunogenicity, selecting adjuvants, establishing vaccine formulation, demonstrating protective efficacy against lethal disease in homologous challenge, achieving full renal clearance using experimental models, and reproducibility of protective efficacy against heterologous challenge. In this review, we highlight the role of the expression/delivery system employed in studies based on the well-known LipL32 and leptospiral immunoglobulin-like (Lig) proteins, as well as the choice of adjuvants, as key factors to achieving the best vaccine performance in terms of protective efficacy against lethal infection and induction of sterile immunity.

Undergraduate laboratory courses are essential to teaching core principles in STEM. This course, Quantitative Biological Methods, provides a unique approach to teaching molecular biology research techniques to students, in a laboratory that is delivered in a sequence that parallels standard biomedical research laboratory protocols. Students attend a lecture where they are taught the essential principles of biomedical research, and a lab where they learn to use laboratory equipment, perform experiments, and purify and quantify DNA and proteins. The course begins with an introduction to laboratory safety, pipetting, centrifugation, spectrophotometry, and other basic laboratory techniques. Next, the lab focuses on the purification and analysis of glutathione S‐transferase (GST) fused to green fluorescent protein (GFP) from an Escherichia coli lysate. Students study this GST‐GFP fusion protein and perform protein quantification, enzyme assays, chromatography, fluorescent detection, normalization, SDS‐PAGE, and western blotting. Students then learn recombinant DNA technology using the GST‐GFP vector that was the source of the fusion protein in the prior labs, and perform ligation, transformation of E. coli cells, blue/white screening, DNA purification via a miniprep, PCR, DNA quantification, restriction enzyme digestion, and agarose gel electrophoresis. Students write laboratory reports to demonstrate an understanding of the principles of the laboratory methods, and they must present and critically analyze their data. The lab methods described herein aim to emphasize the core molecular biology principles and techniques, prepare students for work in a biomedical research laboratory, and introduce students to both GST and GFP, two versatile laboratory proteins. [ABSTRACT FROM AUTHOR]

Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification. [ABSTRACT FROM AUTHOR]

Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.

Proteases are closely associated with many pathological conditions. Efficient detection of protease activity may be useful for diagnosis, prognosis, and the development of new therapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as the non-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins are widely used in FRET biosensors because they can be genetically encoded and compatible with cells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. In this study, it was aimed to detect protease activity using an FPFRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding the mNeonGreen-TEV-mRuby3 fluorescent protein-based FRET biosensor was constructed. The gene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells using recombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed in the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated. [ABSTRACT FROM AUTHOR]

Since antiquity Man has been fascinated by the variations in human (and animal) growth. Stories and art abound about giants and little people. Modern genetics have solved some of etiologies at both extremes of growth. Serious study began with the pathophysiology of acromegaly followed by early attempts at treatment culminating in modern endoscopic surgery and multiple pharmacologic agents. Virtually at the same time experiments with the removal of the pituitary from laboratory animals noted the slowing or stopping of linear growth and then over a few decades the extraction and purification of a protein within the anterior pituitary that restored, partially or in full, the animal’s growth. Human growth hormone was purified decades after those from large animals and it was noted that it was species specific, that is, only primate growth hormone was metabolically active in primates. That was quite unlike the beef and pork insulins which revolutionized the care of children with diabetes mellitus. A number of studies included mild enzymatic digestion of beef growth hormone to determine if those “cores” had biologic activity in primates and man. Tantalizing data showed minimal but variable metabolic efficacy leading to the “active core” hypothesis, for these smaller peptides would be amenable to peptide synthesis in the time before recombinant DNA. Recombinant DNA changed the landscape remarkably promising nearly unlimited quantities of metabolically active hormone. Eight indications for therapeutic use have been approved by the Food and Drug Administration and a large number of clinical trials have been undertaken in multiple other conditions for which short stature in childhood is a sign. The future predicts other clinical indications for growth hormone therapy (and perhaps other components of the GH?IGF-1 axis), longer-acting analogues and perhaps a more physiologic method of administration as virtually all methods at present are far from physiologic.