432The field of recombinant protein production has attained widespread popularity due to the enormous applications of recombinant proteins in therapeutics, diagnostics or to understand basic biological questions. Several factors hamper efficient production of recombinant human proteins in a foreign host. Such aspects are needed to be addressed meticulously in order to increase the expression and yield of the protein while retaining its biological activity. Choice of host system is one of the most critical criterion. Escherichia coli, a Gram-negative bacterium, is a commonly used bacterial host for the production of recombinant proteins. Its use as a cell factory is well-established due to the vast knowledge of its genetics and physiology. Other features include faster doubling time, low-maintenance costs and ability to express proteins in abundance. Hence, several molecular tools and approaches have been developed for enhanced expression and production of heterologous proteins, such as codon optimization soft-wares, a vast catalogue of expression plasmids and engineered strains, and various purification and production strategies. In this review, we discuss these essential prerequisite factors for enhanced expression and yield of human proteins in E. coli, and highlight the biomedical applications of such recombinant proteins in this ever-growing field.
Summary Tumor-driven immune suppression poses a significant impediment to the success of immunotherapy in ovarian cancer. Among the various mechanisms contributing to immune suppression, intracellular communication facilitated by tumor-derived extracellular vesicles (EVs) within the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor growth. We discovered that EVs from both ovarian tumor cell lines and the plasma of ovarian cancer patients are encapsulated with eukaryotic translation initiation factor 4E (eIF4E). Our study revealed a new mechanism showing how these EVs are loaded with eIF4E and its impact on ovarian cancer progression. We also demonstrated that eIF4E-containing EVs (eIF4E-EVs) alter protein translation in macrophages, contributing to anti-tumor immune response. Treatment of macrophages with eIF4E-EVs induces an immunosuppressive phenotype marked by the release of cytokines such as IL-6 and an elevated expression of Programmed death-ligand 1 (PD-L1). Notably, eIF4E-packaged EVs enhance the expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) a pivotal enzyme in cholesterol biosynthesis, resulting in increased cholesterol levels within macrophages. Inhibition of HMGCR or reduction of cholesterol in macrophages effectively restores their antitumor activity by decreasing PD-L1 on macrophages. Analysis of tumor tissue from ovarian cancer patients revealed a positive correlation between HMGCR and TAM in ovarian cancer. In summary, we have characterized the mechanism of how eIF4E loaded EVs induced cholesterol synthesis, creating an immunosuppressive environment by upregulating PD-L1 expression in macrophages.
Undifferentiated transcription factor 1 (UTF1) is a protein found in the nucleus of the cell and is highly conserved and expressed specifically in placental mammals. Its expression in pluripotent and germ cells is primarily associated with cell differentiation, whereas tissue-specific roles (either as a tumor suppressor or as an oncogene) have been observed in the case of cancer cells. Previous reports have exemplified the importance of UTF1 protein for obtaining good quality induced pluripotent stem cells. UTF1 also showed exemplary potential as a biomarker in the identification of these pluripotent cells. Here we report human UTF1 fusion protein expression in E. coli in soluble form and purification using native purification method. The ideal expression parameters were identified and the fusion protein was purified, retaining its secondary structure. UTF1 has dual role either as a tumor suppressor or as an oncogene in cancer paradigm. In cervical cancer cells, we sought to investigate its role in various cellular processes through protein transduction. The results showed UTF1 protein transduction resulted in a reduction in cell proliferation and clonogenic potential of cervical cancer cells. Further, we observed the upregulation in p27Kip1 mRNA levels in the presence of UTF1. The present study provided a promising method to express and purify UTF1 fusion protein from bacterial system and a potent platform to use this biologically active protein to unravel valuable insights with respect to its role in cervical, as well as other cancer paradigms, and induced pluripotent stem cells.
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