TOPO Cloning for Efficient Plasmid Construction.

This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac™ Baculovirus Expression System.

Production of Secreted Antibody in Baculovirus Expression Vector System.

Monoclonal antibodies have widespread applications in disease treatment and antigen detection. They are traditionally produced using mammalian cell expression system, which is not able to satisfy the increasing demand of these proteins at large scale. Baculovirus expression vector system (BEVS) is an attractive alternative platform for the production of biologically active monoclonal antibodies. In this chapter, we demonstrate the production of an HIV-1 broadly neutralizing antibody b12 in BEVS. The processes including transfer vector construction, recombinant baculovirus generation, and antibody production and detection are described.

Scarless Baculovirus Genome Editing Using Lambda-Red Recombineering in E. coli.

Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Optimization of extended Kozak elements enhances recombinant proteins expression in CHO cells.

In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.

Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is a promising candidate for treating metabolic disorder diseases and has been used in phase II clinical trials. Currently, metabolic diseases are prevalent worldwide, underscoring the significant market potential of FGF21. Therefore, the production of FGF21 must be effectively improved to meet market demand. RESULTS: Herein, to investigate the impact of vectors and host cells on FGF21 expression, we successfully engineered strains that exhibit a high yield of FGF21. Surprisingly, the data revealed that vectors with various copy numbers significantly impact the expression of FGF21, and the results showed a 4.35-fold increase in expression levels. Furthermore, the performance of the double promoter and tandem gene expression construction design surpassed that of the conventional construction method, with a maximum difference of 2.67 times. CONCLUSION: By exploring engineered vectors and host cells, we successfully achieved high-yield production of the FGF21 strain. This breakthrough lays a solid foundation for the future industrialization of FGF21. Additionally, FGF21 can be easily, quickly and efficiently expressed, providing a better tool and platform for the research and application of more recombinant proteins.

Expression of Multispecific Antibodies.

Bi- and multispecific antibody formats allow the development of new therapeutic strategies to address previously unmet medical needs. However, due to the increased complexity (e.g., the interface design and the presence of multiple binders), such molecules are generally more challenging to express and purify compared to standard monoclonal antibodies (mAbs). We describe here an optimized methodology to express and purify basic bispecific antibodies using the BEAT® interface. This interface allows to generate antibodies with very high levels of heterodimer product (reported titers exceed 10 g/L) and comes with a built-in purification strategy allowing removal of residual levels of undesired product-related impurities (e.g., homodimers and half molecules).

Expression of VP2 protein of Novel goose parvovirus in baculovirus and evaluation of its immune effect.

Short-beak and dwarfism syndrome (SBDS) is a new disease caused by a genetic variant of goose parvovirus in ducks that results in enormous economic losses for the waterfowl industry. Currently, there is no commercial vaccine for this disease, so it is urgent to develop a safer and more effective vaccine to prevent this disease. In this study, we optimized the production conditions to enhance the expression of the recombinant VP2 protein and identified the optimal conditions for subsequent large-scale expression. Furthermore, the protein underwent purification via nickel column affinity chromatography, followed by concentration using ultrafiltration tube. Subsequently, it was observed by transmission electron microscopy (TEM) that the NGPV recombinant VP2 protein assembled into virus-like particles (VLPs) resembling those of the original virus. Finally, the ISA 78-VG adjuvant was mixed with the NGPV-VP2 VLPs to be prepared as a subunit vaccine. Furthermore, both agar gel precipitation test (AGP) and serum neutralization test demonstrated that NGPV VLP subunit vaccine could induce the increase of NGPV antibody in breeding ducks. The ducklings were also challenged with the NGPV, and the results showed that the maternal antibody level could provide sufficient protection to the ducklings. These results indicated that the use of the NGPV VLP subunit vaccine based on the baculovirus expression system could facilitate the large-scale development of a reliable vaccine in the future.

A low-endotoxic Salmonella enterica Gallinarum serovar delivers infectious bronchitis virus immunogens via a dual-promoter vector system that drives protective immune responses through MHC class-I and -II activation in chickens.

An effective vaccine strategy is indispensable against infectious bronchitis virus (IBV) and fowl typhoid (FT), both of which threaten the poultry industry. This study demonstrates a vector system, pJHL270, designed to express antigens in prokaryotic and eukaryotic cells. The vector system stimulates immune responses via synchronized antigen presentation to MHC class-I and -II molecules to produce balanced Th1/Th2 responses. The vaccine antigens were crafted by selecting the consensus sequence of the N-terminal domain of the spike protein (S1-NTD) and a conserved immunogenic region of the nucleocapsid protein (N321-406 aa) from IBV strains circulating in South Korea. The vaccine antigen was cloned and transformed into a live-attenuated Salmonella Gallinarum (SG) strain, JOL2854 (∆lon, ∆cpxR, ∆rfaL, ∆pagL, ∆asd). Western blot analysis confirmed concurrent antigen expression in Salmonella and eukaryotic cells. Oral immunization with the SG-based IBV vaccine construct JOL2918 induced IBV antigen and Salmonella-specific humoral and cell-mediated immune responses in chickens. PBMCs collected from immunized chickens revealed that MHC class-I and -II expression had increased 3.3-fold and 2.5-fold, respectively, confirming MHC activation via bilateral antigen expression and presentation. Immunization induced neutralizing antibodies (NAbs) and reduced the viral load by 2-fold and 2.5-fold in the trachea and lungs, respectively. The immunized chickens exhibited multifaceted humoral, mucosal, and cell-mediated responses via parallel MHC class-I and -II activation as proof of a balanced Th1/Th2 immune response. The level of NAbs, viral load, and gross and histological analyses provide clear evidence that the construct provides protection against IBV and FT.

Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter via Reverse Genetics.

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in infants and the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, there is a need for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined via Nanopore sequencing, we developed a robust reverse genetics system enabling the recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (~107 plaque-forming units/mL), its growth kinetics were modestly decreased in comparison to the laboratory-adapted OSU virus. The reverse genetics system was used to generate the rOSU rotavirus, which served as an expression vector for a foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced a genetically stable rOSU virus that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse-genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens for other porcine enteric diseases.

Development and application of sugarcane streak mosaic virus vectors.

Sugarcane streak mosaic virus (SCSMV) is one of the major pathogens of sugarcane in the world. Molecular studies and disease management of SCSMV are hindered by the lack of efficient infectious clones. In this study, we successfully constructed Agrobacterium infiltration based infectious clone of SCSMV with different variants. Infectious clones of wild type SCSMV could efficiently infect Nicotiana benthamiana and sugarcane plants resulting in streak and mosaic symptoms on systemic leaves which were further confirmed with RT-PCR and serological assays. SCSMV variants of less adenylation displayed attenuated pathogenicity on N.benthamiana. SCSMV-based recombinant heterologous EGFP protein vector was also developed. The EGFP-tagged recombinant SCSMV could highly expressed in vegetative organs including roots. These infectious clones of SCSMV could be further developed for platform tools for both biotechnological studies and management of SCSMV disease.

From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies.

The increasing demand for biosimilar monoclonal antibodies (mAbs) has prompted the development of stable high-producing cell lines while simultaneously decreasing the time required for screening. Existing platforms have proven inefficient, resulting in inconsistencies in yields, growth characteristics, and quality features in the final mAb products. Selecting a suitable expression host, designing an effective gene expression system, developing a streamlined cell line generation approach, optimizing culture conditions, and defining scaling-up and purification strategies are all critical steps in the production of recombinant proteins, particularly monoclonal antibodies, in mammalian cells. As a result, an active area of study is dedicated to expression and optimizing recombinant protein production. This review explores recent breakthroughs and approaches targeted at accelerating cell line development to attain efficiency and consistency in the synthesis of therapeutic proteins, specifically monoclonal antibodies. The primary goal is to bridge the gap between rising demand and consistent, high-quality mAb production, thereby benefiting the healthcare and pharmaceutical industries.

Novel Enhanced Mammalian Cell Transient Expression Vector via Promoter Combination.

During the emergence of infectious diseases, evaluating the efficacy of newly developed vaccines requires antigen proteins. Available methods enhance antigen protein productivity; however, structural modifications may occur. Therefore, we aimed to construct a novel transient overexpression vector capable of rapidly producing large quantities of antigenic proteins in mammalian cell lines. This involved expanding beyond the exclusive use of the human cytomegalovirus (CMV) promoter, and was achieved by incorporating a transcriptional enhancer (CMV enhancer), a translational enhancer (woodchuck hepatitis virus post-transcriptional regulatory element), and a promoter based on the CMV promoter. Twenty novel transient expression vectors were constructed, with the vector containing the human elongation factor 1-alpha (EF-1a) promoter showing the highest efficiency in expressing foreign proteins. This vector exhibited an approximately 27-fold higher expression of enhanced green fluorescent protein than the control vector containing only the CMV promoter. It also expressed the highest level of severe acute respiratory syndrome coronavirus 2 receptor-binding domain protein. These observations possibly result from the simultaneous enhancement of the transcriptional activity of the CMV promoter and the human EF-1a promoter by the CMV enhancer. Additionally, the synergistic effect between the CMV and human EF-1a promoters likely contributed to the further enhancement of protein expression.

Discovery of novel replication proteins for large plasmids in cyanobacteria and their potential applications in genetic engineering.

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090, both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090-containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090, the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid." Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria.

A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression.

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

Preparation,identification and comparison of murine and rabbit polyclonal antibodies against matrix protein of rabies virus strain CVS-11 / 中国生物制品学杂志

@#Objective To prepare murine and rabbit polyclonal antibodies against rabies virus(RV) matrix(M) protein and compare their reactivity.Methods The prokaryotic expression vector pET-28a-M was constructed by using the cDNA of cells infected with RV CVS-11 strain as template,then transformed into E.coli BL21(DE3),and the induced by IPTG to express M protein.After nickel column affinity chromatography and dialysis renaturation,female BALB/c mice and New Zealand white rabbits were immunized with the M protein,and the whole blood was taken to separate the serum.The titers of the murine and rabbit polyclonal antibodies were detected by ELISA,and the reactivity was measured by Western blot,indirect immunofluorescence assay(IFA) and immunoprecipitation(IP).Results The plasmid pET-28a-M was constructed correctly as identified by sequencing.The titers of murine and rabbit polyclonal antibodies were 1:100 and 1:256 000respectively,and the polyclonal antibodies had reactivity with different RV strains.Conclusion The murine and rabbit polyclonal antibodies against M protein were successfully prepared,which provides important biological tools for exploring the interaction between M protein and host protein as well as studying the pathogenesis of RV.

Recombinant rotavirus expressing the glycosylated S1 protein of SARS-CoV-2.

Reverse genetic systems have been used to introduce heterologous sequences into the rotavirus segmented double-stranded (ds)RNA genome, enabling the generation of recombinant viruses that express foreign proteins and possibly serve as vaccine vectors. Notably, insertion of SARS-CoV-2 sequences into the segment seven (NSP3) RNA of simian SA11 rotavirus was previously shown to result in the production of recombinant viruses that efficiently expressed the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the S1 region of the SARS-CoV-2 spike protein. However, efforts to generate a similar recombinant (r) SA11 virus that efficiently expressed full-length S1 were less successful. In this study, we describe modifications to the S1-coding cassette inserted in the segment seven RNA that allowed recovery of second-generation rSA11 viruses that efficiently expressed the ~120-kDa S1 protein. The ~120-kDa S1 products were shown to be glycosylated, based on treatment with endoglycosidase H, which reduced the protein to a size of ~80 kDa. Co-pulldown assays demonstrated that the ~120-kDa S1 proteins had affinity for the human ACE2 receptor. Although all the second-generation rSA11 viruses expressed glycosylated S1 with affinity for the ACE receptor, only the S1 product of one virus (rSA11/S1f) was appropriately recognized by anti-S1 antibodies, suggesting the rSA11/S1f virus expressed an authentic form of S1. Compared to the other second-generation rSA11 viruses, the design of the rSA11/S1f was unique, encoding an S1 product that did not include an N-terminal FLAG tag. Probably due to the impact of FLAG tags upstream of the S1 signal peptides, the S1 products of the other viruses (rSA11/3fS1 and rSA11/3fS1-His) may have undergone defective glycosylation, impeding antibody binding. In summary, these results indicate that recombinant rotaviruses can serve as expression vectors of foreign glycosylated proteins, raising the possibility of generating rotavirus-based vaccines that can induce protective immune responses against enteric and mucosal viruses with glycosylated capsid components, including SARS-CoV-2.

A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts.

Baculovirus expression system1s are a widely used tool in recombinant protein and biologics production. To enable the possibility of genome modifications unconstrained through low-throughput and bespoke classical genome manipulation techniques, we set out to construct a baculovirus vector (>130 kb dsDNA) built from modular, chemically synthesized DNA parts. We constructed a synthetic version of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) through two steps of hierarchical Golden Gate assembly. Over 140 restriction endonuclease sites were removed to enable the discrimination of the synthetic genome from native baculovirus genomes. A head-to-head comparison of our modular, synthetic AcMNPV genome with native baculovirus vectors showed no significant difference in baculovirus growth kinetics or recombinant adeno-associated virus production-suggesting that neither baculovirus replication nor very-late gene expression were compromised by our design or assembly method. With unprecedented control over the AcMNPV genome at the single-nucleotide level, we hope to ambitiously explore novel AcMNPV vectors streamlined for biologics production and development.