Recombinant DNA Technology Research Articles

HBV Vaccines: Advances and Development.

Hepatitis B virus (HBV) infection is a global public health problem that is closely related to liver cirrhosis and hepatocellular carcinoma (HCC). The prevalence of acute and chronic HBV infection, liver cirrhosis, and HCC has significantly decreased as a result of the introduction of universal HBV vaccination programs. The first hepatitis B vaccine approved was developed by purifying the hepatitis B surface antigen (HBsAg) from the plasma of asymptomatic HBsAg carriers. Subsequently, recombinant DNA technology led to the development of the recombinant hepatitis B vaccine. Although there are already several licensed vaccines available for HBV infection, continuous research is essential to develop even more effective vaccines. Prophylactic hepatitis B vaccination has been important in the prevention of hepatitis B because it has effectively produced protective immunity against hepatitis B viral infection. Prophylactic vaccines only need to provoke neutralizing antibodies directed against the HBV envelop proteins, whereas therapeutic vaccines are most likely needed to induce a comprehensive T cell response and thus, should include other HBV antigens, such as HBV core and polymerase. The existing vaccines have proven to be highly effective in preventing HBV infection, but ongoing research aims to improve their efficacy, duration of protection, and accessibility. The routine administration of the HBV vaccine is safe and well-tolerated worldwide. The purpose of this type of immunization is to trigger an immunological response in the host, which will halt HBV replication. The clinical efficacy and safety of the HBV vaccine are affected by a number of immunological and clinical factors. However, this success is now in jeopardy due to the breakthrough infections caused by HBV variants with mutations in the S gene, high viral loads, and virus-induced immunosuppression. In this review, we describe various types of available HBV vaccines, along with the recent progress in the ongoing battle to develop new vaccines against HBV.

Discovery of molecular and genetic mechanisms of cell cycle regulation: 2001 Nobel laureates leland Hartwell, Timothy Hunt and Paul Nurse

Leland Hartwell, Paul Nurse and Timothy Hunt in 2001 were awarded the Nobel Prize for their discovery of molecular and genetic mechanisms of the cell cycle. The review analyzes the features of the objects chosen by scientists, a wide range of methods from conventional light microscopy to recombinant DNA technology and complementation test, used for research, and covers a history of the discoveries made. Thanks to the work of these scientists, a modern­ understanding of the cell cycle checkpoints, the complexes formed by cyclin and cyclin-dependent­ kinases at different phases of the cell cycle, as well as the mechanism of periodic cyclin degradation and the universality of the cyclin mechanism of cellular division in all living organisms was gained. Keywords: cdc mutants, cdc2/cdc28, cdk protein kinase, cdk/cyclin complex, cell cycle, cyclin, Saccharomyces cere­visiae, Schizosaccharomyces pombe

The advent of plant cells in bioreactors.

Ever since agriculture started, plants have been bred to obtain better yields, better fruits, or sustainable products under uncertain biotic and abiotic conditions. However, a new way to obtain products from plant cells emerged with the development of recombinant DNA technologies. This led to the possibility of producing exogenous molecules in plants. Furthermore, plant chemodiversity has been the main source of pharmacological molecules, opening a field of plant biotechnology directed to produce high quality plant metabolites. The need for different products by the pharma, cosmetics agriculture and food industry has pushed again to develop new procedures. These include cell production in bioreactors. While plant tissue and cell culture are an established technology, beginning over a hundred years ago, plant cell cultures have shown little impact in biotechnology projects, compared to bacterial, yeasts or animal cells. In this review we address the different types of bioreactors that are currently used for plant cell production and their usage for quality biomolecule production. We make an overview of Nicotiana tabacum, Nicotiana benthamiana, Oryza sativa, Daucus carota, Vitis vinifera and Physcomitrium patens as well-established models for plant cell culture, and some species used to obtain important metabolites, with an insight into the type of bioreactor and production protocols.

OP38 Evaluation Of A High-Cost Medicine For A Rare Disease: 16-Year Cohort Of Imiglucerase Use For Gaucher Disease In Brazil

IntroductionGaucher disease is a lysosomal storage disease of autosomal recessive inheritance that is caused by a deficiency of the enzyme glucocerebrosidase. This deficiency results in accumulation of the enzyme’s main substrate in the lysosomes of macrophages, mainly in the spleen, liver, and bone marrow. In more severe cases it can affect the lung, kidneys, and central nervous system. There are two main treatments available for patients with Gaucher disease: enzyme replacement therapy and inhibition of substrate synthesis. The main enzyme replacement therapy used in Brazil is imiglucerase, an analog of the human β-glucocerebrosidase enzyme. Imiglucerase is produced by recombinant DNA technology using a cell culture derived from the Chinese hamster ovary. It has 497 amino acids and differs from the endogenous enzyme by an amino acid at position 495, where histidine is replaced by arginine. The objective of the study was to analyze the survival of patients treated for Gaucher disease with imiglucerase in Brazil from 2000 to 2015.MethodsWe constructed a retrospective cohort study of patients with Gaucher disease who received imiglucerase through the Brazilian National Health System from 2000 to 2015 using a national database created from the linkage of administrative databases.ResultsA total of 1,241 patients who received imiglucerase were included. The overall survival rates at one, ten, and 15 years were 98.7 percent (95% confidence interval [CI]: 98.1, 99.4), 92.3 percent (95% CI: 90.2, 94.4), and 89.4 percent (95% CI: 85.6, 93.3), respectively.ConclusionsOur findings advance the understanding of the profile, survival, and risk factors of people with Gaucher disease, adding new data to the discussion regarding pharmaceutical therapies and patient care, and providing data for the development of new public health policies for the use of advanced, high-cost drugs for rare diseases.

#183 : The Use of Follitropin-Delta in Indonesia: Current Evidence and Development

Background and Aims: The current success of IVF treatments was aided by the fulminant ascent of recombinant DNA technologies. Follitropin delta (FD) is used to promote ovarian follicular proliferation. This research was aimed to investigate the current evidence and development of follitropin delta in its novel in Indonesia. Method: This prospective cohort study was conducted at the Yasmin IVF Center, Dr. Cipto Mangunkusumo General Hospital Jakarta from January 2021 to October 2022. All IVF/ICSI cycles in which follitropin delta (RekovelleⓇ, FE 999049; Ferring Pharmaceuticals, St. Prex, Switzerland) was used for ovarian stimulation. The outcome data were collected, including initial dose, and total dose of follitropin delta, retrieved oocytes, fertilized eggs, blastocyst day 3-5, embryo transfer, clinical pregnancy rates, and clinical implantation rates. The collected patients’ data (n = 20) who underwent IVF and received follitropin delta ovarian stimulation were divided into 2 groups by age (under 35 years and above 35 years). Results: The average oocyte, blastocyst, and embryo day 3 obtained in the study sample were higher in women under 35 years old. The pregnancy rate was higher in group 1 (33,30% vs. 25,0%) which was assessed based on a positive b-HCG results and a P4 value after receiving FET. Patients who followed follitropin delta ovarian stimulation had a pregnancy rate of 30%. Follitropin delta can be used in patients to successfully obtain a pregnancy in both groups, as it achieves higher result in women under 35 years old (33.3% vs. 25%). Conclusion: Optimizing ovarian response in IVF by using a program of follitropin delta dose achieved considerable rise in the quantity of good-quality, usable blastocysts. Assessing follitropin delta in individualized protocols and improving understanding of the dosages can help clinicians in evaluating follitropin delta in their conventional protocols.

Proteomics: Technology, Challenges and Advances

Proteomics is the study of proteins and involves the applications of technologies for the identification and quantification of overall proteins present content of a cell, tissue or an organism. Proteomics is developing fast due to the growing number of proteins obtained by recombinant DNA technology. Proteomics-based technologies are utilized in various capacities for different research settings such as the detection of various diagnostic markers, candidates for vaccine production, understanding pathogenicity mechanisms and interpretation of functional protein pathways in different diseases. Protein analysis employs various techniques and methods, which helps to study the proteomics. While the potential is great, many challenges and issues remain to be solved such as mining and integration of the proteomics. Over the last decade, technological advances in proteomics have driven or increased the capability of proteomics discovery. This review efforts to describe various technologies for the protein analysis, challenges and recent advances employed in the field of proteomics.

Plant Tissue Culture and Formation of Secondary Metabolites - A Review

Many copies of a single plant can be grown using the plant tissue culture technology. These copies have the right characteristics to satisfy medical and nutritional demands. Secondary metabolites are purposefully synthesized by using the in vitro technique. These metabolites act as protectors for plants during stressful conditions and offer resistance against different organisms and factors, ultimately helping the plant to survive. With the passage of time, the development of new instruments for the improved synthesis of secondary metabolites via the genetic control of biosynthetic pathways has been aided by the speedy development of recombinant DNA technology. Plants generate a wide range of secondary metabolites that have various biological functions, such as fungicide, herbicide, anti-parasitic, and anti- microbial functions. Nanotechnology has the potential to drastically alter conventional plant growing methods and bring about the synthesis of flavonoids, anthocyanin, and diosgenin by using silver nanoparticles and cadmium oxide nanoparticles (CdONPs). The technique of callus cultures is increasingly utilized to produce secondary metabolites. Hence, the main objective of the current review is to increase the synthesis of secondary metabolites.

The Quest for Immunity: Exploring Human Herpesviruses as Vaccine Vectors.

Herpesviruses are large DNA viruses that have long been used as powerful gene therapy tools. In recent years, the ability of herpesviruses to stimulate both innate and adaptive immune responses has led to their transition to various applications as vaccine vectors. This vaccinology branch is growing at an unprecedented and accelerated rate. To date, human herpesvirus-based vectors have been used in vaccines to combat a variety of infectious agents, including the Ebola virus, foot and mouth disease virus, and human immunodeficiency viruses. Additionally, these vectors are being tested as potential vaccines for cancer-associated antigens. Thanks to advances in recombinant DNA technology, immunology, and genomics, numerous steps in vaccine development have been greatly improved. A better understanding of herpesvirus biology and the interactions between these viruses and the host cells will undoubtedly foster the use of herpesvirus-based vaccine vectors in clinical settings. To overcome the existing drawbacks of these vectors, ongoing research is needed to further advance our knowledge of herpesvirus biology and to develop safer and more effective vaccine vectors. Advanced molecular virology and cell biology techniques must be used to better understand the mechanisms by which herpesviruses manipulate host cells and how viral gene expression is regulated during infection. In this review, we cover the underlying molecular structure of herpesviruses and the strategies used to engineer their genomes to optimize capacity and efficacy as vaccine vectors. Also, we assess the available data on the successful application of herpesvirus-based vaccines for combating diseases such as viral infections and the potential drawbacks and alternative approaches to surmount them.

PSII-10 Effect of Equine Chorionic Gonadotrophin-Like Glycoprotein Treatment on Fertility in Angus Cows Subjected to a 7-Day Co-Synch Protocol

Abstract This study evaluated the effect of administering a glycoprotein with equine chorionic gonadotropin like activity produced by recombinant DNA technology (eCG-like) associated with a 7-d Co-Synch protocol on estrus rate (ER) and pregnancy per timed AI (P/TAI). A total of 1,344 suckled Angus cows (primiparous, n = 381; multiparous, n = 963) from 11 herds were scanned at initiation of synchronization protocol by transrectal ultrasonography and the presence of corpus luteum (CL) and uterine tone was used to classify them by cycling. On day -10, cows received 100 μg GnRH analogue and a progesterone intravaginal device (IVD; 1.0g progesterone) for 7 days. At IVD removal, cows were administered 500 μg of cloprostenol and randomly assigned to receive 400 IU of eCG-like glycoprotein (Syntex S.A., Argentina, PCT/EP2019/073277; eCG-like, n = 665) or to remain as untreated (Control, n = 679). Also on day -3, paint was applied on the sacrum to the tail head of each cow. Finally, 100 μg GnRH analog were injected concurrently with TAI on Day 0 (66h after IVD removal) and the ER was evaluated by the degree of paint loss. Pregnancy to TAI was determined on day 30 by transrectal ultrasonography when an embryo and fluid were detected in the uterus. Body condition score (BCS; scale = 1-9) was evaluated on days -10 and 30. Optimal BCS was considered ≥ 5 and BCS balance between days -10 and 30 was classified as negative (lost BCS) or positive (maintained or gained BCS). The associations among treatment, parity, cyclicity at initiation of protocol, optimal BCS on day -10, and their interactions and ER and P/TAI were analyzed by GLIMMIX (SAS 9.3). Estrus expression and BCS balance were also included as effects in the model of pregnancy per TAI. The effect of herd was included as a random effect. The model specifications included an option to retrieve the mean, SEM, and an adjustment for multiple comparisons (Tukey-Kramer). The percentage of acyclic cows was 31.3%. The ER in cyclic cows was 64.9% and the eCG-like treatment increased ER only in anestrus cows (55.8 vs. 44.3%; Treatment x cyclicity interaction; P = 0.03). The percentage of cows with negative BCS balance was 27.2%. The P/TAI in cows with positive BCS balance was 48.4% and the eCG-like treatment increased P/TAI only in cows with negative BCS balance (44.7% vs. 30.5%; Treatment x BCS balance interaction; P = 0.01). In conclusion, the addition of a recombinant eCG-like glycoprotein to a 7-day Co-Synch protocol increased estrus expression in anestrus Angus cows and P/TAI in those with negative body condition score balance. However, this addition did not influence the estrus expression in cycling cows or on P/TAI in those with positive BCS balance.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

An Algorithm for the Development of a Recombinant Antiherpetic Subunit Vaccine Combining the Crystal Structure Analysis, AlphaFold2-Based Modeling, and Immunoinformatics

Using the envelope glycoprotein B (gB) crystal structure and digital prediction algorithm, the B- and T-cell antigenic determinants (epitopes) of human herpesvirus 1 (HHV-1), also known as herpes simplex virus 1 (HSV-1), were generated, and the method for their production in the form of recombinant proteins was proposed. First, the structure of the surface topological domain (ectodomain or spike) of gB with mapped epitopes was analyzed, and the most stable and immunogenic (due to their enrichment with B-and T-cell epitopes) subdomains were selected for the modeling of subunit vaccine prototypes using the AlphaFold2 (Google DeepMind, London, UK) artificial intelligence system. The proposed candidate vaccines included both small (about 100 amino acids) monomeric polypeptides, which were ideal for recombinant expression as fusion proteins, and a more complex polypeptide, which, due to its trimeric fold, looks like a miniature analog of the gB ectodomain. In this miniature analog, the ectodomain regions with the potential to interfere efficacious expression of soluble recombinant protein in Escherichia coli have been removed. The structural stability of the modeled proteins, confirmed by molecular dynamics simulation and host immune responses, predicted in silico, indicates the suitability of the two suggested polypeptides for generating subunit vaccines using recombinant DNA technology.

Combined In Silico and In Vitro Study to Reveal the Structural Insights and Nucleotide-Binding Ability of the Transcriptional Regulator PehR from the Phytopathogen Ralstonia solanacearum.

The transcriptional regulator PehR regulates the synthesis of the extracellular plant cell wall-degrading enzyme polygalacturonase, which is essential in the bacterial wilt of plants caused by one of the most devastating plant phytopathogens, Ralstonia solanacearum. The bacterium has a wide global distribution infecting many different plant species, resulting in massive agricultural and economic losses. Because the PehR molecular structure has not yet been determined and the structural consequences of PehR on ligand binding have not been thoroughly investigated, we have used an in silico approach combined with in vitro experiments for the first time to characterize the PehR regulator from a local isolate (Tezpur, Assam, India) of the phytopathogenic bacterium R. solanacearum F1C1. In this study, an in silico approach was employed to model the 3D structure of the PehR regulator, followed by the binding analysis of different ligands against this regulatory protein. Molecular docking studies suggest that ATP has the highest binding affinity for the PehR regulator. By using molecular dynamics (MD) simulation analysis, involving root-mean-square deviation, root-mean-square fluctuations, hydrogen bonding, radius of gyration, solvent-accessible surface area, and principal component analysis, it was possible to confirm the sudden conformational changes of the PehR regulator caused by the presence of ATP. We used an in vitro approach to further validate the formation of the PehR-ATP complex. In this approach, recombinant DNA technology was used to clone, express, and purify the gene encoding the PehR regulator from R. solanacearum F1C1. Purified PehR was used in ATP-binding experiments using fluorescence spectroscopy and Fourier transform infrared spectroscopy, the outcomes of which showed a potent binding to ATP. The putative PehR-ATP-binding analysis revealed the importance of the amino acids Lys190, Glu191, Arg192, Arg375, and Asp378 for the ATP-binding process, but further study is required to confirm this. It will be simpler to comprehend the catalytic mechanisms of a crucial PehR regulator process in R. solanacearum with the aid of the ATP-binding process hints provided by these structural biology applications.

Charge variants of proposed biosimilar to Omalizumab: Isolation, purification and analysis by HPLC methods

Omalizumab (Xolair) is a humanized monoclonal antibody derived by recombinant DNA technology. It binds specifically to immunoglobulin E (IgE) which plays a major role in allergic reaction by releasing histamine and other inflammatory factors from mast cells. Omalizumab binds circulatory IgE with high affinity and prevents from its binding to mast cell receptor. Charge variants are one of the critical quality attributes (CQAs) in biological drug development and sources of heterogeneity which needs to be considered in biosimilarity assessment. In this study, biosimilar product of Xolair was expressed in mammalian cell culture process in laboratory to isolate charge variants (acidic, main peak and basic). Different charge variants were isolated from intermediate purified biosimilar product of Xolair. Isolated charge variants were purified with preparative cation exchange chromatography technique and characterized with different analytical tools includes size exclusion chromatography (SEC-HPLC) and cation exchange chromatography (CEX-HPLC). Purity of acidic, main peak and basic variants was 99.58%, 99.98% and 98.64% respectively as per SEC-HPLC and according to CEX-HPLC purity was 94.25%, 95.58% and 91.33% respectively. The study data indicates that isolated charge variants were purified with desired purity and can be further used for process characterization, in vitro potency and in vivo kinetics studies.

Integrative Molecular Approaches to Plant Disease: A Review

Modern molecular and bioinformatics technologies have made understanding host-pathogen interactions easier. Plants have many ways to protect themselves from microbial diseases, such as physical barriers, PAMP detection, and R genes that recognize pathogen-effector proteins and turn on effector-triggered immunity. Plant pathogen genome databases provide genomic and phenotypic data on plant pathogen species and information on plant-pathogen interactions. Map-based or positional gene cloning is improving our understanding of plant-pathogen interactions, with R genes being used to develop resistance to pathogens. Plant genomes typically contain several hundred nucleotide-binding site-leucine-rich repeats (NLRs), with their number, arrangement, and domain combinations varying by species. Bacterial blight (BB) severely impacts rice production, and about 37 of 44 resistance genes have been mapped and 15 cloned. Many disease-resistant wheat cultivars have been developed using powdery mildew leaf rust (Lr) resistance genes from wild relatives of T. aestivum. Over 140 genes are linked to powdery mildew resistance in T. aestivum and MutChromSeq have found new target genes. Cloning Arabidopsis resistance genes is essential for developing resistant cultivars and understanding R gene evolution. Some R genes encode proteins with nucleotide-binding site (NBS) motifs, and an LRR protects against Erysiphe cruciferarum powdery mildew. CRISPR/Cas9 gene editing is a major tool in plant genome editing, efficiently introducing target site mutations and improving plant immunity. High-throughput sequencing can identify and clone candidate resistance genes in different plant species, and gene editing technologies like CRISPR/Cas have illuminated site-specific mutagenesis and durable resistance.

Advances and Challenges in the Development of Gene Therapy Medicinal Products for Rare Diseases.

The development of viral vectors and recombinant DNA technology since the 1960s has enabled gene therapy to become a real therapeutic option for several inherited and acquired diseases. After several ups and downs in the gene therapy field, we are currently living a new era in the history of medicine in which several ex vivo and in vivo gene therapies have reached maturity. This is testified by the recent marketing authorization of several gene therapy medicinal products. In addition, many others are currently under evaluation after exhaustive investigation in human clinical trials. In this review, we summarize some of the most significant milestones in the development of gene therapy medicinal products that have already facilitated the treatment of a significant number of rare diseases. Despite progresses in the gene therapy field, the transfer of these innovative therapies to clinical practice is also finding important restrictions. Advances and also challenges in the progress of gene therapy for rare diseases are discussed in this opening review of a Human Gene Therapy issue dedicated to the 30th annual Congress of the European Society for Gene and Cell Therapy.

DNAi-peptide nanohybrid smart particles target BCL-2 oncogene and induce apoptosis in breast cancer cells

Genomic DNA sequences provide unique target sites, with high druggability value, for treatment of genetically-linked diseases like cancer. B-cell lymphoma protein-2 (BCL-2) prevents Bcl-2-associated X protein (BAX) and Bcl-2 antagonist killer 1 (BAK) oligomerization, which would otherwise lead to the release of several apoptogenic molecules from the mitochondrion. It is also known that BCL-2 binds to and inactivates BAX and other pro-apoptotic proteins, thereby inhibiting apoptosis. BCL-2 protein family, through its role in regulation of apoptotic pathways, is possibly related to chemo-resistance in almost half of all cancer types including breast cancer. Here for the first time, we have developed a nanohybrid using a peptide-based carrier and a Deoxyribonucleic acid inhibitor (DNAi) against BCL-2 oncogene to induce apoptosis in breast cancer cells. The genetically designed nanocarrier was functionalized with an internalizing RGD (iRGD) targeting motif and successfully produced by recombinant DNA technology. Gel retardation assay demonstrated that the peptide-based carrier binds single-stranded DNAi upon simple mixing. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses further revealed the formation of nanohybrid particles with a size of 30 nm and a slightly positive charge. This hemocompatible nanohybrid efficiently delivered its contents into cancer cells using iRGD targeting moiety. Gene expression analysis demonstrated that the nanohybrids, which contained DNAi against BCL-2 proficiently suppressed the expression of this oncogene in a sequence specific manner. In addition, the nanohybrid, triggered release of cytochrome c (cyt c) and caspase3/7 activation with high efficiency. Although the DNAi and free nanocarrier were separately unable to affect the cell viability, the nanohybrid of 20 nM of DNAi showed outstanding antineoplastic potential, which was adjusted by the ratio of the MiRGD nanocarrier to DNAi. It should be noted that, the designed nanohybrid showed a suitable specificity profile and did not affect the viability of normal cells. The results suggest that this nanohybrid may be useful for robust breast cancer treatment through targeting the BCL-2 oncogene without any side effects.

Soluble Expression of Antimicrobial Peptide BSN-37 from Escherichia coli by SUMO Fusion Technology.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

In-depth mass spectrometry analysis of rhGH administration altered energy metabolism and steroidogenesis

Growth hormone, as a proteohormone, is primarily known of its dramatic effect on longitudinal growth. Recombinant DNA technology has provided a safe, abundant and comparatively cheap supply of human GH for growth hormone-deficient individuals. However, many healthy subjects, especially athletics, administrate GH for enhanced athletic performance or strength. A better and more comprehensive understanding of rhGH effect in healthy individuals is urgent and essential. In this study, we recruited 14 healthy young male and injected rhGH once. Untargeted LC-MS metabolomics profiling of serum and urine was performed before and after the rhGH injection. The GH-induced dysregulation of energy related pathways, such as amino acid metabolism, nucleotide metabolism, glycolysis and TCA cycle, was revealed. Moreover, individuals supplemented with micro-doses of rhGH exhibited significantly changed urinary steroidal profiles, suggesting a role of rhGH in both energy metabolism and steroidogenesis. We expect that our results will be helpful to provide new evidence on the effects of rhGH injection and provide potential biomarkers for rhGH administration.

Strategies for the Development of Industrial Fungal Producing Strains.

The use of microorganisms in industry has enabled the (over)production of various compounds (e.g., primary and secondary metabolites, proteins and enzymes) that are relevant for the production of antibiotics, food, beverages, cosmetics, chemicals and biofuels, among others. Industrial strains are commonly obtained by conventional (non-GMO) strain improvement strategies and random screening and selection. However, recombinant DNA technology has made it possible to improve microbial strains by adding, deleting or modifying specific genes. Techniques such as genetic engineering and genome editing are contributing to the development of industrial production strains. Nevertheless, there is still significant room for further strain improvement. In this review, we will focus on classical and recent methods, tools and technologies used for the development of fungal production strains with the potential to be applied at an industrial scale. Additionally, the use of functional genomics, transcriptomics, proteomics and metabolomics together with the implementation of genetic manipulation techniques and expression tools will be discussed.

The effect of thioredoxin and prochymosin coexpression on the refolding of recombinant alpaca chymosin.

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pET-TrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage.