BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications.
RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity.
CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

BACKGROUND: Oral cancer poses a significant burden on public health in India, with higher incidence and mortality rates. Despite advancements in treatment modalities, prognosis remains poor due to factors such as localized recurrence and lymph node metastasis, potentially influenced by cancer stem cells. Among signaling pathways implicated in CSC regulation, the Hedgehog pathway plays a crucial role in oral squamous cell carcinoma (OSCC).
METHODS: 97 OSCC patients\' tissue samples were collected and subjected to RNA isolation, cDNA synthesis and quantitative real-time PCR to analyze PTCH1 and SMO expression. Protein expression was assessed through immunohistochemistry. Clinicopathological parameters were correlated with gene and protein expression. Statistical analysis included Pearson chi-square tests, co-relation co-efficient tests, Kaplan-Meier survival analysis and ROC curve analysis.
RESULTS: PTCH1 expression correlated with lymphatic permeation (p = 0.002) and tumor stage (p = 0.002), while SMO expression correlated with lymph node status (p = 0.034) and tumor stage (p = 0.021). PTCH1 gene expression correlated with lymph node status (p = 0.024). High PTCH1 gene expression was associated with shorter survival in tongue cancer patients. ROC curve analysis indicated diagnostic potential for PTCH1 and SMO gene and cytoplasmic SMO expression in distinguishing malignant tissues from adjacent normal tissues.
CONCLUSIONS: PTCH1 and SMO play a crucial role in oral cancer progression, correlating with tumor stages and metastatic potential. Despite not directly influencing overall survival, PTCH1 expression at specific anatomical sites hints at its prognostic implications. PTCH1 and SMO exhibit diagnostic potential, suggesting their utility as molecular markers in oral cancer management and therapeutic strategies.

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.

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.

The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.

Thymic epithelial tumors (TETs) are rare and the major symptoms are not obvious until the tumor progresses to a relatively large size and compresses the surrounding organs. As its growth is aggressive and it metastasizes to distant organs, it is important to find novel effective therapies. Lenvatinib, a vascular endothelial growth factor receptor (VEGFR) inhibitor, is approved as a drug therapy for thymic carcinoma (TC); however, although it is a molecular targeted therapy, there are no obvious predictors of therapeutic efficacy. The present study aimed to assess the association between clinicopathological factors and the protein expression of VEGFR, which is associated with tumor aggressiveness and the efficacy of VEGFR inhibitors. The VEGFR-2 protein expression was evaluated in 144 patients with TETs who underwent surgical resection. The present study assessed whether the expression of VEGFR-2 protein was associated with TET classification and pathological stage, progression-free survival and overall survival (OS). A total of 94 cases (65.2%) were positive for VEGFR-2 protein. The expression of VEGFR-2 was higher in the more aggressive type B3 thymoma and TC (88.5%) than in types A, AB, B1 and B2 thymoma (60.2%). The 5-year OS rate for the overall population was 53.1%. The 5-year OS rates of patients with negative VEGFR-2 staining score values (66.5%) were significantly longer than in patients with positive VEGFR-2 staining score values (42.5%; P=0.000078). Furthermore, the pathological stage was the only factor significantly associated with OS in multivariate analysis. The results of the present study suggest the possibility that the indications for VEGF inhibitor therapy could be extended to type B3 thymoma.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

BACKGROUND: Long noncoding RNAs (lncRNAs) are noncoding RNA transcripts greater than 200 nucleotides in length and are known to play a role in regulating the transcription of genes involved in vital cellular functions. We hypothesized the disease process in dysferlinopathy is linked to an aberrant expression of lncRNAs and messenger RNAs (mRNAs).
OBJECTIVE: In this study, we compared the lncRNA and mRNA expression profiles between wild-type and dysferlin-deficient murine myoblasts (C2C12 cells).
METHODS: LncRNA and mRNA expression profiling were performed using a microarray. Several lncRNAs with differential expression were validated using quantitative real-time polymerase chain reaction. Gene Ontology (GO) analysis was performed to understand the functional role of the differentially expressed mRNAs. Further bioinformatics analysis was used to explore the potential function, lncRNA-mRNA correlation, and potential targets of the differentially expressed lncRNAs.
RESULTS: We found 3195 lncRNAs and 1966 mRNAs that were differentially expressed. The chromosomal distribution of the differentially expressed lncRNAs and mRNAs was unequal, with chromosome 2 having the highest number of lncRNAs and chromosome 7 having the highest number of mRNAs that were differentially expressed. Pathway analysis of the differentially expressed genes indicated the involvement of several signaling pathways including PI3K-Akt, Hippo, and pathways regulating the pluripotency of stem cells. The differentially expressed genes were also enriched for the GO terms, developmental process and muscle system process. Network analysis identified 8 statistically significant (P<.05) network objects from the upregulated lncRNAs and 3 statistically significant network objects from the downregulated lncRNAs.
CONCLUSIONS: Our results thus far imply that dysferlinopathy is associated with an aberrant expression of multiple lncRNAs, many of which may have a specific function in the disease process. GO terms and network analysis suggest a muscle-specific role for these lncRNAs. To elucidate the specific roles of these abnormally expressed noncoding RNAs, further studies engineering their expression are required.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac β-myosin heavy chain (β-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.