Foreign Proteins Research Articles

An innovative method of nonclinical efficacy and toxicological evaluation of recombinant Salmonella typhi Ty21a expressing HPV16 and 18 L1 proteins

Advancement in technology led to development of live attenuated Salmonella typhi Ty21a as enteric vector for expression of foreign proteins. Such vector platform is inevitable for development of vaccine candidate against human papilloma virus (HPV), the etiological agent of cervical cancer with high prevalence in developing nations. The high risk HPVs like type 16 and 18 contributes to 70% of cervical cancer, hence Indian Immunologicals Limited (IIL), Hyderabad, India developed a recombinant HPV vaccine by introducing HPV 16 and 18 L1 protein coding genes into attenuated S. typhi Ty21a vector. Being a genetically engineered enteric vector vaccine, it would be less expensive, with an ease of oral administration, instead of injectable that needs trained personale, is an added advantage for low socioeconomic setup compared to existing HPV vaccines.Establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines has become major constrain for such recombinant S. typhi HPV (rSt.HPV) vaccine. Since, the intended clinical mode of rSt.HPV is through oral route, whereas the live attenuated S. typhi Ty21a doesn't colonize in gut of laboratory animals to be used for nonclinical experiments. Hence, an alternate and unconventional method of ‘intranasal drug testing’, was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits administered with rSt.HPV through, intra-peritoneal, intranasal and oral routes, the intended clinical route.•Current unconventional and innovative nonclinical testing procedures helps in exploring the alternate methods by pharmacologist/toxicologist.•Ultimately, such new drugs developed through technology must serve the humankind justifying the guidelines of regulatory agencies.

Composition and Rheological Properties of Peanut Oil Bodies from Aqueous Enzymatic Extraction.

In this study, the relationship between the composition and rheological properties of peanut oil bodies from aqueous enzymatic extraction was evaluated. Aqueous enzymatic extraction using a combination of cellulase and pectinase at a 1:1 ratio effectively destroyed the structure of the cell wall and resulted in the maximum oil body yield of 90.7%. The microstructure and interfacial membrane composition of the peanut oil bodies were observed by confocal laser scanning microscopy. The oil bodies contained three inherent proteins (oleosin, caleosin, and steroleosin) along with two adsorbed foreign proteins (arachin and lipoxygenase). Five phospholipids were detected using 31P nuclear magnetic resonance spectroscopy. Among them, phosphatidylcholine, which plays a major role in the stability of oil bodies, was the most abundant. The measured rheological properties indicated that the oil bodies were a typical elastic system. Elevated temperature and high-speed shear destroyed the binding between proteins and phospholipids, reducing the oil body stability. The findings will facilitate the commercial application of peanut oil bodies by improving the extraction rate of peanut oil bodies and clarifying their stabilization mechanism.Practical Application: This paper studies the enzymatic extraction, composition and rheological properties of peanut oil bodies. It provides a theoretical basis for the large-scale application of peanut oil bodies in the food and cosmetic industries. It is beneficial to improve the application value of peanut resources.

Engineering Chloroplasts for High-Level Constitutive or Inducible Transgene Expression.

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.

Expressing of Recombinant VEGFR2-specific Nanobody in Baculovirus Expression System.

Background: Baculovirus expression system, introduced more than 20 years ago, is considered as a useful tool for large and complex eukaryotic recombinant protein production. A baculovirus expression vector is a recombinant virus which desired foreign protein coding sequences is under control of the virus gene promoter. Baculovirus only infects insect cells and do not normally infect vertebrates therefore, they possess no risk of biological risks for human.Objectives: The aim of this study was to recombinant expression of vascular endothelial growth factor (VEGF) reseptor-2 specific Nanobody in the baculovirus expression systemMaterials and Methods: Gene of specific Nanobody against the VEGF reseptor-2 that called 3VGR19 was cloned and expressed in baculovirus system. Results: 3VGR19 Nanobody gene was amplified by Polymerase Chain Reaction (PCR) using the specific primers, and was cloned in pFastBac HTA plasmid. DH10Bac bacteria was transformed with resulted donor plasmid. The cultured Sf9 insect cell line was transfected with recombinant bacmid, and finally, the expression and purification of 3VGR19 was confirmed in insect cells. Conclusions: In conclusion, Transient infection of insect cells with baculovirus can be a promising technology for expression of antibody fragments.

Protective immune studies against fungi

The immune system is the host's defence against different agents and infections. Understanding the complex and highly dynamic interactions between fungi and host cells in a tissue-specific manner is crucial to facilitate the development of new therapeutic approaches to infections. Generally fungal pathogens rarely cause diseases in immunologically competent individuals. However, commensal and non-pathogenic environmental fungi can cause life-threatening infections in individuals with immune deficiency. Understanding the molecular and cellular bases of immunity to fungi has progressed significantly over the past few years. Despite close interactions with fungi today, how the immune system protects humans and animals from fungal pathogens has not been fully elucidated compared to the immune response to bacteria or viruses. The immune system is the host's defence against various foreign proteins and infections. Understanding the complex and highly dynamic interactions between fungi and host cells is crucial for the development of new therapeutic approaches to infections. Researchers from 15 countries in Europe, Asia, Australia, North and South America have provided the last five years review and original research articles that consist of a wide range of fungal pathogens, disease, effector, regulatory cells and molecular pathways of host immune responses to fungal exposure. In this review, we summarize an outline of the recent findings, perspectives, and reviews about the complex and highly dynamic interactions between fungi and host cells and a contemporary understanding of protective immunity against fungi. This review will allow an overview of the most exciting recent advances in antifungal immunity, discoveries that will help pave the way for the development of new strategies that are seriously needed to combat these devastating diseases.

ალერგია და მისი მკურნალობა ანტიჰისტამინური პრეპარატებით

Allergy is a disease of the 20th century. 30% - 40% of the world's population is allergic to various irritants. "Dangerous" agents invading the human body: bacteria, viruses, airborne dust, animal fur, foreign proteins, etc. cause various allergic diseases and can often lead to serious complications (including lethal outcome). This article discusses the causes and characteristic symptoms of allergies, modern methods of diagnosis and treatment with effective and safe antihistamines - Polesin, Lotegra, Loranex, Erius, Eslotin, Telfast, Allerfast, Nixar, Loratadine, Claritine, Cetek, Alertek , Zetrin, Parlazin, Kestin, Zirtek, Terrix, etc. Immunotherapy - or specific hyposensitization - is also characterized as a modern and effective method of treatment, the principle of which lies in the production of specific tolerance (indiscretion) towards the causative allergen.

Genetically Corrected Autologous Stem Cells Engraft, But Host Immune Responses Limit Their Utility in Canine α-L-iduronidase Deficiency

Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency. Large volume marrow collections were performed on five alpha-ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA. Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs. An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID. Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients. Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion. Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog. We evaluated immune responses against alpha-ID and transduced cells. Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay. Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays. To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A. Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable. In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement. Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins.

Prediction of putative regulatory elements in the subgenomic promoters of cucumber green mottle mosaic virus and their interactions with the RNA dependent RNA polymerase domain.

Characterization of the subgenomic RNA (sgRNA) promoter of many plant viruses is important to understand the expression of downstream genes and also to configure their genome into a suitable virus gene-vector system. Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is one of the RNA viruses, which is extensively being exploited as the suitable gene silencing and protein expression vector. Even though, characters of the sgRNA promoters (SGPs) of CGMMV are yet to be addressed. In the present study, we predicted the SGP for the movement protein (MP) and coat protein (CP) of CGMMV. Further, we identified the key regulatory elements in the SGP regions of MP and CP, and their interactions with the core RNA dependent RNA polymerase (RdRp) domain of CGMMVwas deciphered. The modeled structure of core RdRp contains two palm (1-41 aa, and 63-109 aa), one finger (42-62 aa) subdomains with three conserved RdRp motifs that played important role in binding to the SGP nucleic acids. RdRp strongly preferred the double helix form of the stem region in the stem and loop (SL) structures, and the internal bulge elements. In MP-SGP, a total of six elements was identified; of them, the affinity of binding to - 26nt to - 17nt site (CGCGGAAAAG) was higher through the formation of strong hydrogen bonds with LYS16, TYR17, LYS19, SER20, etc. of the motif A in the palm subdomain of RdRp. Similar strong interactions were noticed in the internal bulge (CAACUUU) located at + 33 to + 39 nt adjacent to the translation start site (TLSS) (+ 1). These could be proposed as the putative core promoter elements in MP-SGP. Likewise, total five elements were predicted within - 114nt to + 144 nt region of CP-SGPwith respect to CP-TLSS. Of them, RdRp preferred to bind at the small hairpin located at - 60 nt to - 43 nt (UUGGAGGUUUAGCCUCCA) in the upstream region, and at the complex duplex structure spanning between + 99 and + 114 nt in the downstream region, thus indicating the distribution of core promoter within - 60 nt to + 114 nt region of CP-SGPwith respect to TLSS (+ 1)of the CP; whereas, the - 114 nt to + 144 nt region of CP-SGP might be necessary for the full activity of the CP-SGP. Ourinsilicoprediction certifies the gravity of these nucleotide stretches as the RNA regulatory elements and identifies their potentiality for binding with of palm and finger sub-domain of RdRp. Identification of such elements will be helpful to anticipate the critical length of the SGPs. Our finding will not only be helpful to delineate the SGPs of CGMMV but also their subsequent application in the efficient construction of virus gene-vector for the expression of foreign protein in plant.

Тransplastomic tobacco plants producing the hydrophilic domain of the sheep pox virus coat protein L1R

Sheep pox has a wide geographical range of distribution and poses a threat to sheep breeding worldwide, as the disease is highly contagious and is accompanied by large economic losses. Vaccines based on live attenuated virus strains are currently being used for prevention of this disease. Such vaccines are effective, but potentially dangerous because of the possible virus reversion to a pathogenic state. The development of safe recombinant subunit vaccines against sheep pox is very relevant. The high ploidy level of the plant chloroplasts makes it possible to obtain large quantities of foreign proteins. The purpose of this study was to create transplastomic Nicotiana tabacum plants producing one of the candidate vaccine proteins of sheep pox virus L1R. A vector containing a deletion variant of the SPPV_56 gene, which encodes the N-terminal hydrophilic part of the viral coat protein L1R, was constructed to transform tobacco plastids. It provides integration of the transgene into the trnG/trnfM region of the chloroplast tobacco genome by homologous recombination. Spectinomycin-resistant tobacco lines were obtained by biolistic gun-mediated genetic transformation. PCR analysis in the presence of gene-specific primers confirmed integration of the transgene into the plant genome. Subsequent Northern and Western blot analysis showed the gene expression at the transcriptional and translational levels. The recombinant protein yields reached up to 0.9 % of total soluble protein. The transplastomic plants displayed a growth retardation and pale green leaf color compared to the wild type, but they developed normally and produced seeds. Southern blot analysis showed heteroplasmy of the plastids in the obtained plants due to recombination events between native and introduced regulatory plastid DNA elements. The recombinant protein from plant tissue was purified using metal affinity chromatography. Future research will be focused on determining the potential of the chloroplast-produced protein to induce neutralizing antibodies against SPPV strains.

Development of immunogenic chimeric virus-like particles based on bovine papillomavirus type 6

Virus-like particles (VLPs) are considered useful tools for vaccine development because they induce an immune response and are safe. In addition, VLPs may be useful as a platform for the presentation of foreign antigens to elicit immune responses. In this study, we aimed to produce a chimeric VLP composed of L1 protein of bovine papillomavirus type 6 (BPV6-L1) that can display an entire foreign protein on its surface. Based on prediction of the conformational structure of VLP of BPV6-L1 (BPV6-VLP), candidate insertion sites for the foreign protein into BPV6-VLP were identified. Fusion proteins of BPV6-L1 and EGFP as a model foreign protein were constructed and produced. Only the fusion protein in which EGFP was inserted between amino acids 136 and 137 of BPV6-L1 self-assembled into VLPs and did not exhibit hindrance of the conformation of EGFP. Chimeric BPV6-VLP-immunized mice produced specific IgG against both BPV6 and EGFP. This is the first demonstration of the production of an immunogenic, bivalent, chimeric BPV6-VLP incorporating an entire protein for stable surface display. Thus, immunogenic chimeric BPV6-VLP may constitute a promising vaccine platform.

Research Progress of the UPR Mechanism and its Effect on Improving Foreign Protein Expression.

The unfolded protein response (UPR) is a protective mechanism against endoplasmic reticulum (ER) stress that induces a series of signal transduction pathways to eliminate misfolded proteins. The UPR mechanism is highly conserved in fungi, higher organisms, plants and mammals. The UPR pathway is activated to stabilize ER functions when there are too many unfolded proteins or misfolded proteins in the ER. However, stress continues when ER proteins are stimulated by toxic substances that affect the balance of the UPR pathway, which causes changes in the structure and function of the ER and other organelles. These ultimately disrupt homeostasis in the body and cause pathological reactions that can be fatal. The UPR mechanism has clear effects on stabilizing the protein-folding environment. Dysfunction or disruption of the UPR mechanism is associated with numerous disorders, including neurodegenerative diseases, loss of control of protein secretion, cerebral ischemia and epilepsy, neuropsychiatric diseases, eye diseases, skin diseases, metabolic and inflammatory diseases, atherosclerosis, and heart disease. Thus, characterization of UPR function and its dysfunction has significant importance and has broad application prospects, which make research into the UPR a research hotspot.

Manipulatingearly pig embryos

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a longterm preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demiembryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been bom upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and < 10% develop to morulae or blastocysts in vitro. Pig oocytes can be stimulated parthenogenetically and up to 10% grow to blastocysts in the in vitro culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind considerable amounts of foreign DNA preferably at the post-acrosomal region, suggesting that transgenic animals can be obtained more efficiently than with the usual microinjection procedure. To increase gene transfer efficiency, considerable research efforts have been made to establish embryonic stem (ES) cells, but so far there is no definite proof of totipotency of the generated pig ESlike cells through viable chimaeras. In general, biotechnological procedures are much less advanced in pigs than in cows.

Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells

The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.

Engineering therapeutic antibodies for patient safety: tackling the immunogenicity problem.

Established monoclonal antibodies (mAbs) allow treatment of cancers, autoimmune diseases and other severe illnesses. Side effects either arise due to interaction with the target protein and its biology or result from of the patient's immune system reacting to the foreign protein. This immunogenic reaction against therapeutic antibodies is dependent on various factors. The presence of non-human sequences can trigger immune responses as well as chemical and post-translational modifications of the antibody. However, even fully human antibodies can induce immune response through T cell epitopes or aggregates. In this review, we briefly describe, how therapeutic antibodies can interact with the patient's immune system and summarize recent advancements in protein engineering and in silico methods to reduce immunogenicity of therapeutic monoclonal antibodies.

CRISPR/Cas technology promotes the various application of Dunaliella salina system.

Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells.

C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.

Articles of Significant Interest in This Issue

Rotaviruses are a leading cause of severe diarrheal disease in young children throughout the world. Using reverse genetics technologies, Philip and Patton (e00959-20) found that the rotavirus RNA genome is remarkably flexible and can accommodate additional coding sequence sufficient to allow expression of separate foreign proteins. Among the recombinant rotaviruses that were engineered are those that produce fluorescent reporters, enabling the study of rotavirus biology and pathogenesis by live cell imaging. Given the capacity of rotavirus to encode foreign proteins, the potential exists for developing rotavirus vaccines as vector systems against other enteric and mucosal diseases.

Fusion with matrix attachment regions enhances expression of recombinant protein in human HT-1080 cells

Like endogenous proteins, recombinant foreign proteins produced in human cell lines also need post-translational modifications. However, high and long-term expression of a gene of interest (GOI) presents significant challenges for recombinant protein production in human cells. In this work, the effect of human matrix attachment region elements (MARs), including the β-globin MAR (gMAR), chicken lysozyme MAR (cMAR), and a combination of these two, on the stable expression of GOI was assessed in human HT-1080cells. After transfection with vectors containing the MAR elements and eGFP, stably HT-1080cell pools were obtained under selective pressure. eGFP protein expression was analyzed by flow cytometry, while transgene copy number and eGFP mRNA expression levels were determined with qPCR and qRT-PCR technology. We found that MARs could not enhance transfection efficiency, but gMAR could significantly increase eGFP expression in stable HT-1080cell pools by approximately 2.69-fold. Moreover, gMAR could also increase eGFP expression stability during long-term culture. Lastly, we showed that the effect of the MARs on transgenes was related to the gene copy number. In summary, this study found that MARs could both enhance the transgene expression and stability in HT-1080cells.

Development of a novel enhanced baculovirus expression vector via promoter combination

Abstract Baculovirus expression systems (BESs) have been used to express many foreign proteins. BESs are known to be very suitable for foreign protein production due to the strong gene expression ability of the polyhedrin promoter. However, expression of foreign proteins using the polyhedrin promoter begins at a very late phase, in the last stage of viral infection. This causes insufficient production of foreign proteins due to death of the host cells. Therefore, our study aimed to construct a vector that can enable not only earlier production of foreign recombinant proteins but also increased production. For this purpose, a novel overexpression vector based on the polyhedrin promoter was constructed by using transcriptional enhancers (hr3 and repeated burst sequences (BSs)) and an additional promoter capable of inducing earlier gene expression than the polyhedrin promoter. Among the vectors with additional promoters that were evaluated, the overexpression vector with the p6.9 promoter showed the highest foreign protein expression efficiency. This vector showed approximately 94 times greater and one-day-earlier expression of the foreign protein than the control vector containing only the polyhedrin promoter. In order of greatest to least influence, the factors that increased the expression efficiency of the polyhedrin promoter were the repeated BSs, the p6.9 promoter and hr3.

A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.