Multigene Expression System Research Articles

Development of a multigene expression system using 2A peptides in Rhodosporidium toruloides.

In eukaryotes, gene expression typically requires individual promoter and terminator for each gene, making the expression of multiple genes tedious and sometimes too difficult to handle. This is especially true for underdeveloped nonmodel organisms with few genetic engineering tools and genetic elements such as Rhodosporidium toruloides. In contrast, polycistronic expression offers advantages such as smaller size and ease of cloning. Here we report the development of a multigene expression system using 2A peptides in R. toruloides. First, twenty-two 2A peptides were evaluated for their cleavage efficiencies, which ranged from 33.65% to 93.32%. Subsequently, the 2A peptide of ERBV-1 with the highest efficiency was selected to enable simultaneous expression of four proteins. In addition, we demonstrated the optimization of the α-linolenic acid biosynthetic pathway using ERBV-1 peptide mediated polycistronic expression, which increased the α-linolenic acid production by 104.72%. These results suggest that using ERBV-1 peptide is an efficient strategy for multigene expression in R. toruloides.

Development of a 2A peptide-based multigene expression system and its application for enhanced production of ganoderic acids in Ganoderma lucidum

Ganoderma has received much attention for its medicinal value, but the manipulation of multiple genes remains a challenge, hindering the genetic engineering of this species for the development of cell factories. Here, we first showed that the presence of an intron is necessary for the efficient expression of the endogenous cDNA of carboxin-resistant gene (cbx) in G. lucidum. Then, the self-cleaving function of 2 A peptide was investigated in G. lucidum by linking cbx cDNA to the codon-optimized hygromycin B-resistant gene (ophph) using the 2A-peptide sequence. The results showed that cbx cDNA and ophph can be successfully expressed in G. lucidum in a bicistronic manner from a single transcript. Moreover, the expression of both genes was not affected by the order within the 2 A cassette. In addition, simultaneous expression of cbx cDNA, ophph, and codon-optimized yellow fluorescent protein gene (opyfp) was conducted for the first time in G. lucidum using the 2 A peptide-based approach. The developed method was successfully applied to express both cDNA of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and squalene epoxidase gene (se) for enhanced production of ganoderic acids (GAs) in G. lucidum. The engineered strain produced the maximum content of GA-Mk, GA-T, GA-S, and GA-Me were 26.56±3.53,39.58±3.75, 16.54±2.16, and 19.1±1.87 μg/100 mg dry weight, respectively. These values were 3.85-, 4.74-, 3.65-, and 3.23-fold higher than those produced by the control strain. The developed method will be useful for the manipulation of complex metabolic or regulatory pathways involving multiple genes in Ganoderma.

Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

A Multi-plex Protein Expression System for Production of Complex Enzyme Formulations in Trichoderma reesei.

Historically, heterologous protein production has been challenging using the hyper-cellulolytic fungus, Trichoderma reesei. T. reesei is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for identification of successful transformants. In this work, we have applied the 2A-peptide multi-gene expression system to co-express four enzymes, which include three cellulases, a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1); as well as the enhanced Green Fluorescent Protein, (eGFP), used here as a marker for monitoring expression levels. We designed a new chassis vector, pTrEno-4X-2A for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each of the cellulases was assessed using pNP glycosides and the chromogenic substrate, AZCL-HE-cellulose, which confirmed the functionality of the enzymes. Expression and activity of these enzymes was proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. Although all three cellulase proteins were successfully expressed, their expression levels varied significantly. Specifically, up to an 18-fold difference was observed between the first and the third gene within the 2A-peptide construct, based on protein quantitation. The availability of this new screening tool is expected to greatly impact multi-enzyme applications, such as production of complex commercial enzyme formulations and the study of metabolic pathway enzymes, especially those destined for cell free applications.

Modular Inducible Multigene Expression System for Filamentous Fungi.

Inducible promoters are indispensable elements when considering the possibility to modulate gene expression on demand. Desirable traits of conditional expression systems include their capacity for tight downregulation, high overexpression, and in some instances for fine-tuning, to achieve a desired product's stoichiometry. Although the number of inducible systems is slowly increasing, suitable promoters comprising these features are rare. To date, the concomitant use of multiple regulatable promoter platforms for controlled multigene expression has been poorly explored. This work provides pioneer work in the human pathogenic fungus Aspergillus fumigatus, wherein we investigated different inducible systems, elucidated three candidate promoters, and proved for the first time that up to three systems can be used simultaneously without interfering with each other. Proof of concept was obtained by conditionally expressing three antifungal drug targets within the ergosterol biosynthetic pathway under the control of the xylose-inducible PxylP system, the tetracycline-dependent Tet-On system, and the thiamine-repressible PthiA system. IMPORTANCE In recent years, inducible promoters have gained increasing interest for industrial or laboratory use and have become key instruments for protein expression, synthetic biology, and metabolic engineering. Constitutive, high-expressing promoters can be used to achieve high expression yields; however, the continuous overexpression of specific proteins can lead to an unpredictable metabolic burden. To prevent undesirable effects on the expression host's metabolism, the utilization of tunable systems that allow expression of a gene product on demand is indispensable. Here, we elucidated several excellent tunable promoter systems and verified that each can be independently induced in a single strain to ultimately develop a unique conditional multigene expression system. This highly efficient, modular toolbox has the potential to significantly advance applications in fundamental as well as applied research in which regulatable expression of several genes is a key requirement.

Potential Proteins Interactions with Bombyx mori Nucleopolyhedrovirus Revealed by Co-Immunoprecipitation.

Simple SummaryThe Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the major pathogens of the silkworm. We reconstituted the virus in vitro and used it as a bait for immunoprecipitation experiments on cells and silkworm bodies, obtaining a database of proteins potentially interacting with BmNPV. This study provides a protein data reference for the screening of BmNPV receptors and the study of proteins that play a key role in the replication of BmNPV. It is also important to deal with the prevention of BmNPV in silkworms at the molecular level.Virus–host interactions are critical for virus replication, virulence, and pathogenicity. The Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the most common and harmful pathogens in sericulture. Herein, we used co-immunoprecipitation to identify candidate proteins with potential interactions with BmNPV. First, a recombinant BV virus particle rBmBV-egfp-p64-3×flag-gp64sp was constructed using a MultiBac baculovirus multigene expression system. Co-immunoprecipitation experiments were then performed with the recombinant BV virus infected with BmN cells and Dazao silkworms. LC-MS/MS analysis revealed a total of 845 and 1368 candidate proteins were obtained from BmN cells and silkworm samples, respectively. Bioinformatics analysis (Gene Ontology, KEGG Pathway) was conducted for selection of proteins with significant enrichment for further confirmation of the effects on BmNPV replication. Overall, the results showed that SEC61 and PIC promoted the replication of BmNPV, while FABP1 inhibited the replication of BmNPV. In summary, this study reveals the potential proteins involved in BmNPV invasion and proliferation in the host and provides a platform for identifying the potential receptor proteins of BmNPV.

Abstract 3557: Toward a personalized off-the-shelf cellular immunotherapy for cancer

Abstract Objectives: We have been developing SV-BR-1-GM, a breast cancer cell line with features of an antigen presenting cell which has been stably transfected with the CSF2 gene encoding GM-CSF. SV-BR-1-GM has been in clinical trials in a regimen including low-dose pre-dose cyclophosphamide to reduce immune suppression, and post-dose local interferon alpha to boost the immune response. The SV-BR-1-GM regimen has been administered alone or in combination with PD-1 inhibitors in patients with advanced breast cancer. We have noted that patients that match the SV-BR-1-GM cell line at least at 1 HLA allele are more likely to derive clinical benefit. Therefore, steps were taken to genetically modify the SV-BR-1 cell line to match more patients. Methodology: We focused on the least polymorphic HLA types in the population: HLA-A (Class I) and HLA-DRB3/4/5 (Class II). The published allele frequencies (Gragert 2013) for HLA-A and HLA-DRB3/4/5 were evaluated for the major demographic groups in the US. The following HLA alleles were selected: A*02:01, A*01:01, A*03:01, A*24:02, A*11:01, A*68:01, A*23:01, A*33:03 for Class I and DRB4*01:01, DRB3*02:02, DRB3*01:01, DRB5*01:01, DRB3*03:01, DRB5*01:02, DRB5*02:02 for HLA-DRB3/4/5. Based on population analysis, this combination of alleles should produce at least a single match in~ 99% of the population, with a ~92% match at Class I HLA-A alleles and a ~98% match at Class II HLA-DRB3/4/5 alleles. SV-BR-1 was modified using CRISPR technology deleting expression of the endogenous HLA-A and HLA-DRB3 alleles. Four lentiviral vectors were constructed to express the HLA alleles, along with the CSF2 gene (which encodes GM-CSF), using a 2A self-cleaving multi-gene expression system. Each lentiviral vector expressed 4 HLA types: 2 HLA-A and 2 HLA-DRB3/4/5 types. Preliminary Data: Following sequential CRISPR treatment, the SV-BR-1 cells were cloned, and one clone selected (clone 17) for further engineering. Lack of expression of HLA-A and HLA-DRB3 was confirmed using flow cytometry, and DNA sequencing. Clone 17 was subsequently transduced separately with the four lentiviral vectors each expressing four HLA genes as well as the CSF2 gene. Following selection and cloning, clones were evaluated by ELISA, flow cytometry and RT-PCR to confirm gene expression. Several clones that secreted GM-CSF and expressed both Class I and Class II HLA alleles have subsequently been transferred to GMP manufacturing. These modified breast cancer cell lines will be used in clinical studies designed to first evaluate the safety of intradermal inoculation with the irradiated cells and later combined with other agents to augment the immune response. Each patient will be treated with the cell line that matches them at least at one HLA allele. Gragert L, et. al. Hum Immunol. 2013;74(10):1313-1320. Citation Format: William V. Williams, Markus D. Lacher, Vivek G. Sunkari, Charles Wiseman, Miguel Lopez-Lago. Toward a personalized off-the-shelf cellular immunotherapy for cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3557.

Characterization of virus-like particles assembled by co-expression of BmCPV capsid shell protein and large protrusion protein

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the Reoviridae family of the Cypovirus genus. Previous studies have shown that the BmCPV major capsid shell protein (CSP) has the ability to self-assemble into virus-like particles (VLPs), and cryo-electron microscopy of the BmCPV virions has revealed a tight mutual binding region between CSP and another capsid protein known as the Large Protrusion Protein (LPP), which further stabilizes the capsid shell. In this study, the multi-gene baculovirus expression system, Ac-MultiBac, was used to produce both solely CSP-based and CSP-LPP co-assembled VLPs. Transmission electron microscopy (TEM) results showed that addition of LPP did not affect the assembly of VLPs resulting in almost identical structure in both cases. However, ex vivo administration of VLPs to silkworm midgut tissue showed that CSP-based VLPs did not induce a significant transcriptional response in the innate immunity and RNAi gene cascades, compared to the co-assembled CSP-LPP based VLPs and the natural BmCPV virions isolated from polyhedra. The experimental results indicate that CSP and LPP attach tightly (“Plug and Display” model with CSP acting as “catcher” and LPP as “tag”) to form VLPs that have a structure similar to that of the native CPV virions. Moreover, our results showed that the formation of VLPs with the two BmCPV capsid proteins is feasible, which can form the basis for the production of BmCPV-based VLPs as a new type of biological material to display exogenous proteins.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the genus Cypovirus in the family Reoviridae. The results of cryo-electron microscopy showed that the BmCPV capsid consists of 60 asymmetric units, and each asymmetric unit contains one turret protein (TP), two large protrusion proteins (LPP) and two capsid shell proteins (CSP). CSP has the ability to self-assemble into virus-like particles (VLPs), and the small protrusion domain (SPD) in CSP may play an essential role in the assembly of viral capsids. In this study, three critical amino acid sites, D828, S829 and V945, in the SPD were efficiently mutated (point mutation) based on the principle of PCR circular mutagenesis. Moreover, a multi-gene expression system, Ac-MultiBac baculovirus, was used to produce eight different recombinant VLPs in vitro. Transmission electron microscopy showed that the single site and double site mutations had little effect on the efficiency and morphology of the assembly of VLPs. Still, the simultaneous mutation of the three sites had a significant impact. The experimental results demonstrate that the SPD of CSP plays an essential role in assembly of the viral capsid, which lays the foundation for further analysis of the molecular and structural mechanism of BmCPV capsid assembly.

Multigene engineering of medium-chain fatty acid biosynthesis in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system.

Medium-chain fatty acids (MCFAs) are an ideal feedstock for biodiesel and a range of oleochemical products. In this study, different combinations of CnFATB3, CnLPAAT-B and CnKASI from coconut (Cocos nucifera L.) were coexpressed in transgenic Arabidopsis thaliana by a Cre/LoxP multigene expression system. Transgenic lines expressing different combinations of these genes were designated FL (FatB3 + LPAAT-B), FK (FatB3 + KASI) and FLK (FatB3 + LPAAT-B + KASI). The homozygous seeds of transgenic Arabidopsis thaliana expressing high levels of these genes were screened, and their fatty acid composition and lipid contents were determined. Compared with its content in wild-type A. thaliana, the lauric acid (C12:0) content was significantly increased by at least 395%, 134% and 124% in FLK, FL and FK seeds, respectively. Meanwhile, the myristic acid (C14:0) content was significantly increased by at least 383%, 106% and 102% in FL, FLK and FK seeds, respectively, compared to its level in wild-type seeds. Therefore, the FLK plants exhibited the best effects to increase the level of C12:0, and FL expressed the optimal combination of genes to increase the level of 14:0 MCFA.

Rational Design of Flavonoid Production Routes Using Combinatorial and Precursor-Directed Biosynthesis.

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.

A novel Cre-enabled tetracycline-inducible transgenic system for tissue-specific cytokine expression in the zebrafish: CETI-PIC3.

ABSTRACTMaladaptive signaling by pro-inflammatory cytokines (PICs), such as TNFα, IL1β and IFNɣ, can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed Cre-enabled and tetracycline-inducible transgenic system for conditional, tissue-specific expression of pro-inflammatory cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of proteins Tumor necrosis factor a (Tnfa), Interleukin-1 beta (Il1b) and Interferon gamma (Ifng1), and H2B-GFP fluorescent reporter in a dose-dependent manner. Furthermore, cytokine misexpression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction demonstrated increased expression levels of tnfa, il1b and ifng1 mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both Tnfa immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species-mediated stress and endoplasmic reticulum stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis and therapy development of diseases with an underlying inflammatory etiology.This article has an associated First Person interview with the first author of the paper.

Expression of recombinant apopholasin using a baculovirus–silkworm multigene expression system and activation via dehydrocoelenterazine

Pholasin is a photoprotein derived from the glowing bivalve mollusk, Pholas dactylus. Even though the chemical structure of the prosthetic group (chromophore) responsible for the light emission character of the mollusk remains unknown, research has shown that the presence of dehydrocoelenterazine (DCL) increased light emission and that the dithiothreitol adduct of DCL was isolated from Pholasin®. To date, our research has been focused on activating apopholasin, the naturally occurring apoprotein of Pholasin®, using DCL. In the current study, the expression of recombinant apopholasin via a baculovirus–silkworm multigene expression system is reported. Additionally, the purification of apopholasin using a Flag®-affinity column, the activation of apopholasin using DCL, and the initiation of its luminescent character through the addition of a peroxidase–hydrogen peroxide mixture are reported. The peroxidase–H2O2-dependent luminescence was observed from the recombinant apopholasin activated with DCL.

A construction strategy for a baculovirus-silkworm multigene expression system and its application for coexpression of type I and type II interferons.

The Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus expression system (BES) is a eukaryotic expression system. It possesses great capability for post‐translation modification in expression of foreign proteins. With the counterselection cassette rpsL‐neo and phage λ‐Red recombinase, the defective‐rescue BmNPV BES reBmBac can be employed for efficient heterologous multigene coexpression at different gene sites in one baculovirus genome. In the present study, a recombinant baculovirus, reBm‐Cαγ, carrying two types of chicken interferon (IFN) genes (chIFN‐α and chIFN‐γ) was constructed using the reBmBac system. The chIFN‐α and chIFN‐γ genes were inserted into the same baculovirus genome at the polyhedron and p10 gene sites, respectively. The recombinant baculovirus was capable of coexpressing both chIFN‐α and chIFN‐γ. The expression levels of the two types of IFN in the coexpression product were exponentially high, at approximately 1.7 and 2.5 times higher, respectively, than those in the corresponding single‐expression products. The increase in expression level corresponds to replacement of the nonessential p10 gene in the reBm‐Cαγ recombinant baculovirus. This coexpression of recombinant chicken IFNs showed superior antiviral activity.

Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm.

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Baculovirus-based genome editing in primary cells

Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).

Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system.

Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure. About 1mg/larva human type II collagen is purified from silkworm skin, which shows a putative large scale of collagen production way. An activity assay of recombinant human type II collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. Scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the baculovirus-silkworm multigene expression system can be used as an efficient platform for express active human type II collagen and other complicated eukaryotic proteins.

2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori.

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues.

Production of novel value-added compounds in transgenic crops has become an increasingly viable approach in recent years. However, in many cases, product yield still falls short of the levels necessary for optimal profitability. Determination of the limiting factors is thus of supreme importance for the long-term viability of this approach. A significant challenge to most metabolic engineering projects is the need for strong coordinated co-expression of multiple transgenes. Strong constitutive promoters have been well-characterized during the >30 years since plant transformation techniques were developed. However, organ- or tissue-specific promoters are poorly characterized in many cases. Oilseeds are one such example. Reports spanning at least 20 years have described the use of certain seed-specific promoters to drive expression of individual transgenes. Multi-gene engineering strategies are often hampered by sub-optimal expression levels or improper tissue-specificity of particular promoters, or rely on the use of multiple copies of the same promoter, which can result in DNA instability or transgene silencing. We describe here a flexible system of plasmids that allows for expression of 1-7 genes per binary plasmid, and up to 18 genes altogether after multiple rounds of transformation or sexual crosses. This vector system includes six seed-specific promoters and two constitutive promoters. Effective constitutive and seed-specific RNA interference gene-suppression cloning vectors were also constructed for silencing of endogenous genes. Taken together, this molecular toolkit allows combinatorial cloning for multiple transgene expression in seeds, vegetative organs, or both simultaneously, while also providing the means to coordinately overexpress some genes while silencing others.