Dataset Information

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

ABSTRACT:

Background

The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae.

Results

A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins.

Conclusions

The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.

SUBMITTER: Sinah N

PROVIDER: S-EPMC3527174 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Publications

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

Sinah Namita N Williams Charlotte A CA Piper Robert C RC Shields S Brookhart SB

BMC biotechnology 20120823

<h4>Background</h4>The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli an ...[more]

PMID: 22916790

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Project description:Nucleotide-activated effector deployment, prototyped by interferon-dependent immunity, is a common mechanistic theme shared by immune systems of several animals and prokaryotes. Prokaryotic versions include CRISPR-Cas with the CRISPR polymerase domain, their minimal variants, and systems with second messenger oligonucleotide or dinucleotide synthetase (SMODS). Cyclic or linear oligonucleotide signals in these systems help set a threshold for the activation of potentially deleterious downstream effectors in response to invader detection. We establish such a regulatory mechanism to be a more general principle of immune systems, which can also operate independently of such messengers. Using sensitive sequence analysis and comparative genomics, we identify 12 new prokaryotic immune systems, which we unify by this principle of threshold-dependent effector activation. These display regulatory mechanisms paralleling physiological signaling based on 3'-5' cyclic mononucleotides, NAD+-derived messengers, two- and one-component signaling that includes histidine kinase-based signaling, and proteolytic activation. Furthermore, these systems allowed the identification of multiple new sensory signal sensory components, such as a tetratricopeptide repeat (TPR) scaffold predicted to recognize NAD+-derived signals, unreported versions of the STING domain, prokaryotic YEATS domains, and a predicted nucleotide sensor related to receiver domains. We also identify previously unrecognized invader detection components and effector components, such as prokaryotic versions of the Wnt domain. Finally, we show that there have been multiple acquisitions of unidentified STING domains in eukaryotes, while the TPR scaffold was incorporated into the animal immunity/apoptosis signal-regulating kinase (ASK) signalosome.IMPORTANCE Both prokaryotic and eukaryotic immune systems face the dangers of premature activation of effectors and degradation of self-molecules in the absence of an invader. To mitigate this, they have evolved threshold-setting regulatory mechanisms for the triggering of effectors only upon the detection of a sufficiently strong invader signal. This work defines general templates for such regulation in effector-based immune systems. Using this, we identify several previously uncharacterized prokaryotic immune mechanisms that accomplish the regulation of downstream effector deployment by using nucleotide, NAD+-derived, two-component, and one-component signals paralleling physiological homeostasis. This study has also helped identify several previously unknown sensor and effector modules in these systems. Our findings also augment the growing evidence for the emergence of key animal immunity and chromatin regulatory components from prokaryotic progenitors.

Project description:AIM:To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/Flag, and to express HBsAg S, MS, and LS proteins in SP2/0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably. METHODS:Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind III/Not I followed by ligation with pRc/CMV, or BamHI/EcoR I followed by ligation with pSG5UTPL/Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSG5UTPL/Flag plasmid vectors with T4 DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were Compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0- pRc/CMV-MS clones were established through clone screening with G418. RESULTS:Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmed to be S, preS2S, preS1-preS2S and preS1S encoding genes, determined by sequencing. The results of Western-blot hybridization were positive for the anticipated proteins. Among them, pRc/CMV-S or pRc/CMV-MS demonstrated the highest expressing their respective antigen. CONCLUSION:Eight recombinant plasmids expressing S, M, L or preS1S proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells, the vector pRc/CMV is superior to pSG5UTPL/Flag, and pRc/CMV-S and pRc/CMV-MS are the most efficient in the pRc/CMV clones. SP2/0 cells stably expressing HBsAg are established, and may be used as target cells for evaluating the CTL activity of a DNA vaccine in vitro.

Dataset Information

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

Background

Results

Conclusions

Publications

A set of dual promoter vectors for high throughput cloning, screening, and protein expression in eukaryotic and prokaryotic systems from a single plasmid.

Similar Datasets

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