Insect Expression Systems Research Articles

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

Functional evaluation of CYP2C19 and CYP3A4 gene polymorphism on ibuprofen metabolism

AimIbuprofen is the most commonly used analgesic. CYP polymorphisms are mainly responsible for the differences in drug metabolism among individuals. Variations in the ability of populations to metabolize ibuprofen can lead to drug exposure events. The aim of this study was to evaluate the effects of CYP2C19 and CYP3A4 polymorphisms on ibuprofen metabolism in a Chinese population. MethodsFirst, 31 CYP2C19 and 12 CYP3A4 microsomal enzymes were identified using an insect expression system. Then, variants were evaluated using a mature incubation system. Moreover, ibuprofen metabolite content was determined via ultra-performance liquid chromatography-tandem mass spectrometry analysis. Finally, kinetic parameters of CYP2C19 and CYP3A4 genotypes were determined via Michaelis-Menten curve fitting. ResultsMost variants exhibited significantly altered intrinsic clearance compared to the wild type. In the CYP2C19 metabolic pathway, seven variants exhibited no significant alterations in intrinsic clearance (CLint), six variants exhibited significantly high CLint (121–291%), and the remaining 15 variants exhibited substantially reduced CLint (1–71%). In the CYP3A4 metabolic pathway, CYP3A4*30 was not detected in the metabolite content due to the absence of activity, and 10 variants exhibited significantly reduced CLint. ConclusionTo the best of our knowledge, this is the first study to assess the kinetic characteristics of 31 CYP2C19 and 12 CYP3A4 genotypes on ibuprofen metabolism. However, further studies are needed on poor metabolizers as they are more susceptible to drug exposure. Our findings suggest that the kinetic characteristics in combination with artificial intelligence to predict the toxicity of ibuprofen and reduce any adverse drug reactions.

Linear epitopes on the capsid protein of norovirus commonly elicit high antibody response among past-infected individuals

BackgroundHuman norovirus (HuNoV) is the leading cause of acute nonbacterial gastroenteritis globally, and its infection is usually self-limited, so most people become past Norovirus (NoV)-infected individuals. It is known that some antibody responses may play a critical role in preventing viral infection and alleviating disease; however, the characteristics and functions of particular antibody responses in persons with previous infections are not fully understood. Capsid proteins, including VP1 and VP2, are crucial antigenic components of NoV and may regulate antibody immune responses, while epitope-specific antibody responses to capsid proteins have not been comprehensively characterized.MethodsWe prepared purified VP1 and VP2 proteins by ion exchange chromatography and measured serum antigen-specific IgG levels in 398 individuals by ELISA. Overlapping 18-mer peptides covering the full length of VP1 and VP2 were synthesized, and then we identified linear antigenic epitopes from 20 subjects with strong IgG positivity. Subsequently, specific antibody responses to these epitopes were validated in 185 past infected individuals, and the conservation of epitopes was analyzed. Finally, we obtained epitope-specific antiserum by immunizing mice and expressed virus-like particles (VLPs) in an insect expression system for a blockade antibody assay to evaluate the receptor-blocking ability of epitope-specific antibodies.ResultsThe IgG responses of VP1 were significantly stronger than those of VP2, both of which had high positive rates of over 80%. The overall positive rate of VP1-IgG and/or VP2-IgG was approximately 94%, which may be past NoV-infected individuals. Four linear antigenic B-cell epitopes of capsid proteins were identified, namely, VP1199–216, VP1469–492, VP297–120, and VP2241–264, all of which were conserved. The IgG response rates of the above epitopes in past NoV-infected individuals were 38.92%, 22.16%, 8.11% and 28.11%, respectively. In addition, VP1199–216- and VP1469–492-specific antibodies can partially block the binding of VLPs to the receptor histo-blood group antigen (HBGA).ConclusionThis is the first study to describe specific antibody responses of VP2 and to identify its B-cell epitopes. Our findings offer data for a more thorough understanding of norovirus capsid protein-specific IgG responses and could provide useful information for designing and developing vaccines.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

N-glycosylation modification of heat shock protein gp96 affects its immunological function

N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System.

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.

昆虫杆状病毒基因转移与表达系统在海洋动物中的应用进展

昆虫杆状病毒基因转移与表达系统在海洋动物中的应用进展

Cloning, Expression and Purification of Recombinant Forms of Full Length and Extracellular Domain EBOV Glycoprotein within Mammalian Cell-Lines

Background: Full length Ebolavirus glycoprotein (GP) intersperses the outer most lipid membrane to form spikes, where it mediates virus-host cell interaction. A secretory form of GP (sGP) is also produced by all 5 known Ebolavirus species. These attributes make GP an ideal target for research and development (R and D) of Ebolavirus and possibly pan-filovirus targeted Rapid Diagnostic Tests (RDTs), bio-therapeutics and vaccines. Prior cloning of recombinant Zaire ebolavirus (EBOV) GP has majorly used insect (baculovirus ) expression systems. We report the cloning, expression and purification of the full length and extracellular domain (ECD) forms of recombinant EBOV GP in mammalian cell-lines. Methods and results: 2034 and 1956 base-pair (bp) coding DNA sequences corresponding to the 669 and 643 amino acids (aa) residues of full length and ECD forms of EBOV GP were sub-cloned into the pTGE plasmids. Recombinant pTGE-plasmids were used to transfect 293-6E HEK mammalian cells grown in serum-free FreeStyleTM 293 Expression Medium. Cell lysates and or culture supernatants were used to obtain purified protein, followed by analysis on SDS-PAGE and Western blot. Purified full length GP was detected as membrane bound protein in cell lysates with estimated molecular weight of ~100 kDa (Cal.M.W.~71.67 kDa) on Western blot; and 0.02 mg GP (Concentration: 0.2 mg/mL, Purity: ~50%) derived. On the contrary, ECD GP was detected in supernatants of cell culture broth with estimated molecular weights of ~116 kDa based on SDS-PAGE and Western blot; and 1.6 mg (Concentration: 0.4 mg/ml, Purity: ~70%) of GP_ECD was obtained. Conclusion: Within mammalian cells, recombinant full length EBOV GP is predominantly expressed as transmembrane protein (tGP), while ECD GP is eluted into the culture medium. Both recombinant forms of GP are critical for the R and D of rapid diagnostic tests (RDTs).

Evaluation of viral contamination in a baculovirus expression system.

Insect expression systems based on baculovirus are widely used for generating recombinant proteins. Here, the infectivity of baculoviruses under the physiological stresses of 'freeze-thaw' and sonication and the baculoviral contamination of recombinant proteins after protein purification were evaluated. Our findings suggest that Nonidet P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses. We therefore suggest that baculovirus is completely inactivated by NP-40 treatment and that recombinant proteins are unlikely to be contaminated with infectious baculoviruses after their affinity purification.

In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6.

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.

Therapeutic Effect of Exogenous Truncated IK Protein in Inflammatory Arthritis.

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562, a leukemia cell line. It is known to be an inhibitory regulator of interferon-γ-induced major histocompatibility complex class (MHC) II expression. Previously, we found that transgenic (Tg) mice constitutively expressing truncated IK (tIK) showed reduced numbers of pathogenic Th1 and Th17 cells, which are known to be involved in the development of rheumatoid arthritis (RA). Here, we investigated whether exogenous tIK protein has a therapeutic effect in arthritis in disease models and analyzed its mechanism. Exogenous tIK protein was produced in an insect expression system and applied to the collagen antibody-induced arthritis (CAIA) mouse disease model. Injection of tIK protein alleviated the symptoms of arthritis in the CAIA model and reduced Th1 and Th17 cell populations. In addition, treatment of cultured T cells with tIK protein induced expression of A20, a negative regulator of nuclear factor-κB (NFκB)-induced inflammation, and reduced expression of several transcription factors related to T cell activation. We conclude that exogenous tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action to biopharmaceutical agents, such as tumor necrosis factor antagonists, that are currently used to treat RA.

Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis.

Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Å resolution and were indexed in space group P1, with unit-cell parameters a = 41.9, b = 48.1, c=62.2 Å, α = 97.3, β = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å3 Da-1 and a solvent content of 50.97%.

Development of a rapid diagnostic kit for antigen and antibody of Middle East respiratory syndrome coronavirus

Abstract In 2015, there was an outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea. Therefore, rapid diagnostic kits for the viral antigen and antibodies against MERS-CoV are urgently required. First, we produced and purified a spike nanoparticle of MERS-CoV by the insect expression system. It was highly soluble and intact for immunization. The spike nanoparticle was used as a coating antigen for enzyme-linked immunosorbent assay (ELISA) to detect MERS-CoV-specific antibodies in the serum. The spike nanoparticle, at least with 1.5 ng/well as a coating antigen, can detect serially diluted standard antibodies purified from rabbits immunized with inactivated MERS-CoV, and from rats immunized with spike nanoparticle. Moreover, it can successfully detect MERS-CoV in patient serum. Next, to develop sandwich ELISA to detect MERS-CoV antigens, we generated 616 hybridoma clones and selected 37 monoclonal antibodies (MAbs), which exclusively reacted against the spike nanoparticle. Among them, three MAbs (S-15, S-16, and S-34) showed about 40% plaque reduction against MERS-CoV. Of the 37 MAbs, we selected the two that showed the optimal combinations to develop sandwich ELISA. This sandwich ELISA also can detect serially diluted MERS-CoV antigen. In this study, we developed two MERS-CoV-specific ELISA kits. One is for detecting MERS antibodies in patient serum and the other is for detecting MERS-CoV itself. Both ELISA kits can be reliably used for diagnosis of MERS-CoV.

Exogenous truncated IK protein ameliorates inflammatory arthritis.

Abstract The truncated IK (tIK) cytokine is known for its ability to downregulate the major histocompatibility complex class II on antigen-presenting cells and to regulate immune activation in inflammatory diseases by suppressing differentiation of pathogenic T cells and activation of macrophage. Because of these immune regulatory functions, previous research has proposed tIK as a new candidate for autoimmune disease therapy such as that for rheumatoid arthritis (RA). However, its immunological mechanism is still not identified clearly. In this study, we showed that exogenous tIK protein produced by the insect expression system suppressed the differentiation of TH1 and TH17 cells, which are key factors of inflammatory arthritis. We also showed that tIK protein reduced symptoms of the disease in the collagen antibody-induced arthritis (CAIA) mouse model. In addition, to investigate the receptor and signal transducers that directly interact with tIK and/or the intracellular molecules as a signal pathway, we performed a protein–protein interaction array. According to protein array data, FAM104B, CUTA, GSN, YEATS4 and some other proteins might interact with tIK protein. Among them some proteins may serve as the negative regulators of NF-kappaB transcription factor. Therefore, we suggest that tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action compared with the currently-used biologics for RA, such as monoclonal antibody drugs.

Expression, purification and characterization of inactive and active forms of ERK2 from insect expression system

Extracellular signal-regulated kinase 2 (ERK2) is a serine/threonine protein kinase involved in many cellular programs, such as cell proliferation, differentiation, motility and programed cell-death. It is therefore considered an important target in the treatment of cancer. In an effort to support biochemical screening and small molecule drug discovery, we established a robust system to generate both inactive and active forms of ERK2 using insect expression system. We report here, for the first time, that inactive ERK2 can be expressed and purified with 100% homogeneity in the unphosphorylated form using insect system. This resulted in a significant 20-fold yield improvement compared to that previously reported using bacterial expression system. We also report a newly developed system to generate active ERK2 in insect cells through in vivo co-expression with a constitutively active MEK1 (S218D S222D). Isolated active ERK2 was confirmed to be doubly phosphorylated at the correct sites, T185 and Y187, in the activation loop of ERK2. Both ERK2 forms, inactive and active, were well characterized by biochemical activity assay for their kinase function. Inactive and active ERK2 were the two key reagents that enabled successful high through-put biochemical assay screen and structural drug discovery studies.

An Overview and History of Glyco-Engineering in Insect Expression Systems.

Insect systems, including the baculovirus-insect cell and Drosophila S2 cell systems are widely used as recombinant protein production platforms. Historically, however, no insect-based system has been able to produce glycoproteins with human-type glycans, which often influence the clinical efficacy of therapeutic glycoproteins and the overall structures and functions of other recombinant glycoprotein products. In addition, some insect cell systems produce N-glycans with immunogenic epitopes. Over the past 20 years, these problems have been addressed by efforts to glyco-engineer insect-based expression systems. These efforts have focused on introducing the capacity to produce complex-type, terminally sialylated N-glycans and eliminating the capacity to produce immunogenic N-glycans. Various glyco-engineering approaches have included genetically engineering insect cells, baculoviral vectors, and/or insects with heterologous genes encoding the enzymes required to produce various glycosyltransferases, sugars, nucleotide sugars, and nucleotide sugar transporters, as well as an enzyme that can deplete GDP-fucose. In this chapter, we present an overview and history of glyco-engineering in insect expression systems as a prelude to subsequent chapters, which will highlight various methods used for this purpose.

Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.

Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

AbstractColorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.