Dataset Information

Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

ABSTRACT: Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being released into culture media. We further determined that the beta-barrel structure and net negative charges of sfGFP played important roles in its auto-extracellular secretion property. Using sfGFP as a carrier, heterologous proteins ranging from peptide to complex protein, including antibacterial peptide PG4, endo-beta-N-acethylglucosamindase H (Endo H), human arginase-1 (ARG1), and glutamate decarboxylase (GAD) were all successfully expressed and secreted extracellularly when fused to the carboxyl end of sfGFP. Besides facilitating the extracellular secretion, sfGFP fusion proteins can also be correctly folded and formed the active complex protein structure, including the trimetric human ARG1 and homo-hexametric GAD. This is the first report that sfGFP can guide the secretion of recombinant proteins out of the cells from cytoplasm in E. coli without affecting their conformation and function.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC5539203 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

Zhang Zhen Z Tang Rongxing R Zhu Dewu D Wang Wenfeng W Yi Li L Ma Lixin L

Scientific reports 20170801 1

Protein secretion in Escherichia coli is usually led by a signal peptide that targets the protein to specific secretory pathways. In this study, we demonstrated that the superfolder green fluorescent protein (sfGFP) could be served as a non-signal peptide to guide protein auto-secretion in E. coli. This auto-secretion was characterized as a three-step process through the sub-cellular localization analysis: inner membrane trans-location followed by anchoring at outer membrane, and then being rele ...[more]

PMID: 28765554

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Project description:UnlabelledWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.ImportanceAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Project description:The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24 °C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24 °C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Dataset Information

Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

Publications

Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli.

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