Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis

Yang, Hui; Li, Shihao; Li, Fuhua; Yu, Keming; Fang, Yang; Xiang, Jianhai

doi:10.3390/md14080152

Cited by 26 publications

(7 citation statements)

References 50 publications

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“…ALFPm3 from black tiger shrimp P. monodon [ 54 ] showed antifungal and antibacterial activities. Bacterial cell membranes have been destroyed by mFcALF2 (modified ALF from Fenneropenaeus chinensis ) resulting in the leakage of cytoplasm [ 55 ]. MjALF-D from Marsupenaeus japonicas also exhibited antibacterial activity [ 4 ].…”

Section: Discussionmentioning

confidence: 99%

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

Anju

Archana

Anooja

et al. 2023

Journal of Genetic Engineering and Biotechnology

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Background Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. Results This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. Conclusion Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

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Section: Discussionmentioning

confidence: 99%

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

Anju

Archana

Anooja

et al. 2023

Journal of Genetic Engineering and Biotechnology

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show abstract

“…Then, the PCR, using 5′ AOX1 and 3′ AOX1 as the primer pair, and using genomic DNA as a template, was performed to validate the positive transformants. The correct transformants were selected for further studies [ 21 ].…”

Section: Methodsmentioning

confidence: 99%

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Wang

Sheng

et al. 2022

Molecules

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Autolysis is a common physiological process in eukaryotic cells that is often prevented or applied, especially in yeast expression systems. In this study, an antimicrobial peptide from chicken (AMP) was recombinantly expressed in the Pichia pastoris expression system, which induced a series of cellular autolysis phenotypes after methanol treatment, such as the aggregated, lysed, irregular, and enlarged cell morphology, while the cells expressing a recombinant aflatoxin-detoxifizyme (ADTZ) were not autolyzed. A comparative transcriptomic analysis showed that the transcriptomic profiles of cells derived from the autolysis and non-autolysis groups were well discriminated, suggesting that the mechanisms of autolysis were at the transcriptional level. A further differential expression gene (DEG) analysis showed that the DEGs from the two groups were involved mainly in autophagy, the MAPK signaling pathway, transcriptional factors, the central carbon metabolism, anti-stress functions, and so on. In the autolysis group, the cell activity was significantly reduced with the MAPK signaling pathway, the central carbon metabolism was down-regulated, and components of the cytoplasm-to-vacuole targeting (CVT) and mitophagy pathways were up-regulated, suggesting that the autophagy involved in the trafficking of intracellular molecules in the vacuole and mitochondrion contributed to autolysis, which was regulated by transcriptional factors and signal pathways at the transcriptional level. This study provides a theoretical basis for genetic modifications to prevent or utilize cell autolysis in the recombinant protein expression system.

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“…Following centrifugation at 10,000 × g for 20 min at 4°C, the supernatant was collected and the protein concentration was quantified by a BCA assay. Total protein (40 µg per lane) was used for western blot analyses and was probed with primary antibodies specific to SOCS1, pSTAT1 and STAT1 according to a protocol described previously ( 16 ).…”

Section: Methodsmentioning

confidence: 99%

Silencing of suppressor of cytokine signaling 1 enhances the immunological effect of mucin 1-calreticulin-primed 4T1 cell-treated dendritic cells in breast cancer treatment

et al. 2017

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In cancer immunotherapy, dendritic cell (DC)-based vaccines represent a promising, yet challenging, treatment method. In addition to overcoming the low expression levels of antigenic epitopes on cancer cells, it is also necessary to overcome the inhibitory effect of suppressor of cytokine signaling 1 (SOCS1) on DC self-antigen presentation. Our group previously demonstrated that calreticulin (CRT) translocated type I transmembrane glycoprotein mucin 1 (MUC1), a breast cancer antigen, to the surface of 4T1 cells, and that treatment with MUC1-CRT-primed 4T1 cell-treated DCs induced apoptosis in a breast cancer cell line. In the present study, cell penetrate peptide, hpp10-DRBD was successfully used to deliver siRNAs into bone marrow-derived (BM) DCs to construct SOCS1-silenced DCs, which were incubated with MUC1-CRT-primed 4T1 cells, and antigen-specific antitumor immunity was markedly enhanced in vitro and in vivo. These results demonstrated that SOCS1-silencing, combined with MUC1-CRT-primed 4T1 cell treatment, may induce increased cytokine production and T cell proliferation by DCs. Furthermore, the in vivo experimental data demonstrated that the silencing of SOCS1 combined with MUC1-CRT-primed 4T1 treatment of BMDCs may induce enhanced immunological effects. The results of the present study have implications for the development of more effective DC-based tumor vaccines, suggesting that the combination of high tumor-associated antigen expression levels on cancer cells with the silencing of a critical inhibitor of DC antigen presentation may be beneficial.

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Recombinant Expression of a Modified Shrimp Anti-Lipopolysaccharide Factor Gene in Pichia pastoris GS115 and Its Characteristic Analysis

Cited by 26 publications

References 50 publications

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression

The Transcriptomic Mechanism of a Novel Autolysis Induced by a Recombinant Antibacterial Peptide from Chicken Expressed in Pichia pastoris

Silencing of suppressor of cytokine signaling 1 enhances the immunological effect of mucin 1-calreticulin-primed 4T1 cell-treated dendritic cells in breast cancer treatment

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