The Type I Interferon cytokine family member, Interferon-α2b (hIFN-α2b), modulates a number of important biological mechanisms including anti-proliferation, immunoregulation and antiviral responses. ...Due to its role in the immune system, hIFN-α2b has been used as a therapeutic modulator in hepatitis C as well as some forms of leukaemia. Clinical grade hIFN-α2b is typically produced in bacterial expression systems that involves complex refolding protocols and subsequent loss of yields. In this study, we describe an expression and purification system for hIFN-α2b from mammalian cells. Application of the Trypsin-1 signal peptide-propeptide domain significantly improved the expression and secretion of hIFN-α2b from HEK293 cells. We established a simple purification strategy that yields homogenous, pure hIFN-α2b that is stable and biologically active.
•Heterologous signal peptide engineering improved recombinant IFN-α2b secretion.•Purified IFN-α2b from mammalian cells was thermostable, monomeric and functional.•The purified IFN-α2b was biologically active and comparable to E. coli-derived material.
Current immunoassays for herbicide detection are usually based on polyclonal or monoclonal antibodies (MAbs) raised in animals. The mammalian expression system allows the procurement of specific and ...highly sensitive antibodies, avoiding animal immunization. In this study, S-metolachlor-specific IgG vectors bearing either Thosea asigna virus 2A or internal ribosome entry site (S-T2A or S-IRES) and single-chain variable fragment (scFv) vectors were designed and expressed. The recombinant antibodies (RAbs) were characterized by indirect competitive enzyme-linked immunosorbent assays (icELISA). The results showed that full-length RAbs exhibited significantly better performance than scFv, and both bicistronic vectors expressed antibodies of correct size, while RAb S-T2A elicited a higher yield than RAb S-IRES. Further analyses showed that RAb S-T2A and RAb S-IRES exhibited comparable reactivities and specificities to the parental MAb, with IC50 values of 3.44, 3.89 and 3.37 ng/mL, respectively. Finally, MAb- and RAb-based icELISAs were established for the determination of S-metolachlor in environmental waters. The recoveries were in the range of 73.0–128.1%, and the coefficients of variation were mostly below 10%. This article describes the production of RAbs for S-metolachlor from mammalian cells for the first time and paves the way to develop RAb-based immunoassays for monitoring herbicide residues in the environment.
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•A mammalian expression system was established to produce RAbs specific for S-metolachlor.•The antibody performance of scFv, and two full-length antibodies bearing T2A or IRES were compared.•The prepared full-length RAbs showed high affinity that preserved the parental MAb properties.•The sensitive and reliable icELISAs were developed based on full-length RAbs.•The developed icELISAs were applied for determination of S-metolachlor in environmental waters.
Spring viraemia of carp virus (SVCV), a highly pathogenic fish rhabdovirus, caused an acute hemorrhagic and highly contagious disease in cyprinids. In this study, a new strain of SVCV was isolated ...from diseased zebrafish in Yangling, Shaanxi Province, China, and named SVCV-CG01. Tissue filtrates of diseased fish were detected by Nest PCR using the specific 714 bp and 606 bp fragments from the glycoprotein gene of SVCV. The genes encoding for the N, P, M and G protein were amplified, and the fragments were inserted into pMD-19T vectors to determine the genome of SVCV. Based on phylogenetic analysis of G protein gene, the isolated virus was classified into the Ia genogroup. The titer of the new strain at 48 h and 72 h was approximately 108 and 109 times higher than that of the 0504 strain, respectively. The virus could produce typical cytopathic effects (CPE) and induce apoptosis in EPC cells at 48 and 72 h post infection (hpi). Healthy zebrafish were injected with different doses of SVCV and mass mortality of fish was detected after 4 d infection, with a cumulative death rate of 100% from 107 TCID50 group at 7 d post infection (dpi). Using cultured mammalian 293T cells, an average transfection efficiency of the four structural proteins was 49.1% and 69.6% at 24 and 48 h post transfection (hpt), respectively. This study could be served as a reference for the research on the molecular epidemiology, evolution and pathogenicity of fish viruses.
•A novel highly virulent SVCV was isolated and identified from diseased zebrafish.•The high pathogenicity of SVCV (CG-01) was observed in vitro and in vivo.•The structural proteins of CG-01 strain could be highly expressed in 293T cells.
On the immune cell surface, many immune receptors are expressed and modulate the inhibitory or activating signals to control the immune responses. Recently, some of these receptors have been ...categorized as immune checkpoint receptors and targeted for cancer immunity or autoimmune diseases. To analyze the weak and fast binding typical for immune receptor-ligand interactions, a real-time surface plasmon resonance (SPR) technique is useful. However, it sometimes becomes difficult to optimize the immobilization conditions and appropriate controls. Considering that receptor orientation is relevant for achieving function on the cell surface, it is important to immobilize ligand proteins using specific tags at the membrane proximal end to avoid steric hindrance and structural changes in specific binding regions. Here we introduce a sensor chip, Sensor Chip CAP (Cytiva), which enables reversible and orientation-controlled immobilization of biotinylated ligands, resulting in a significant cost-effective method. We further show preparation methods of several biotinylated immune receptor proteins for SPR analysis, which are also useful for structural and other functional analyses.
•Stable recombinant ZIKV NS1-His-expressing HEK293 cells were generated.•Rapamycin treatment followed by serum starvation leads to a 29-fold increase in recombinant ZIKV NS1 protein secretion.•The ...purified recombinant ZIKV NS1 hexamer is a reliable biological tool for clinical diagnosis and surveillance purposes.
Sensitive, accurate and cost-effective diagnostic tests are urgently needed to detect Zika virus (ZIKV) infection. Nonstructural 1 (NS1) glycoprotein is an excellent diagnostic marker since it is released in a hexameric conformation from infected cells into the patient's bloodstream early in the course of the infection. We established a stable rZNS1-His-expression system in HEK293 cells through lentiviral transduction. A novel optimization approach to enhance rZNS1-His protein secretion in the mammalian expression system was accomplished through 50 nM rapamycin incubation followed by serum-free media incubation for 9 days, reaching protein yields of ∼10 mg/l of culture medium. Purified rZNS1-His hexamer was recognized by anti-NS1 antibodies in ZIKV patient's serum, and showed the ability to induce a humoral response in immunized mice. The obtained recombinant protein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to detect ZIKV in infected patients during the acute phase.
Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the ...characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22–25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22–30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d.
There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock‐down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA.
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The design of gRNA is crucial for the selection of CRISPR/Cas13 system. Our study shows that 22‐25 nt sgRNA with Cas13b and 22‐30 nt sgRNA with Cas13d can play a good knock‐down role by targeting the middle region of gene transcript. It is easy to downregulate genes in mice by expressing Cas13 in different ways, this allows Cas13b and Cas13d to show great potential for RNA manipulation and disease treatment in vivo without damaging genomic DNA.
Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant ...proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model “difficult-to-express” virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90β, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other “difficult-to-express” virus capsid proteins in the mammalian cell system.
•Preliminary exploration of the mechanism underlying the limited expression of PCV2 capsid protein in HEK293F cells.•The engineered PCV2 capsid protein could be spontaneously assemble into nanoparticles with an estimated mean diameter of 41 nm.•An integrated strategy of protein engineering combined with VPA addition promoted the expression of PCV2 capsid protein.
Mammalian cells are widely used for producing recombinant glycoproteins of pharmaceutical interest. However, a major drawback of using mammalian cells is the high production costs associated with ...uniformly isotope-labeled glycoproteins due to the large quantity of labeled
l
-glutamine required for their growth. To address this problem, we developed a cost-saving method for uniform isotope labeling by cultivating the mammalian cells under glutamine-free conditions, which was achieved by co-expression of glutamine synthase. We demonstrate the utility of this approach using fucosylated and non-fucosylated Fc glycoforms of human immunoglobulin G1.
Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a ...challenge as the novelty and increased complexity of new targets often makes them ‘difficult‐to‐express’. This study aimed to define the molecular features that restrict the production of a model ‘difficult‐to‐express’ recombinant protein, Tissue Inhibitor Metalloproteinase‐3 (TIMP‐3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass ‘secretory’ bottlenecks and result in efficient recombinant protein production.
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