Lineage Tracing for the Study and Manipulation of Heterogeneous Cell Populations
Danielle
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Here, we describe the Control of Lineages by Barcode Enabled Recombinant Transcription (COLBERT) method in which unique single guide RNA (sgRNA) barcodes are used as functional tags to identify and recall specific lineages of interest.
An sgRNA barcode is stably integrated and actively transcribed, such that all cellular progeny will contain the parental barcode and produce a functional sgRNA. The sgRNA barcode has all the benefits of a DNA barcode and added functionalities. Once a barcode pertaining to a lineage of interest is identified , the lineage of interest can be isolated using an activator variant of Cas9 (such as dCas9-VPR) and a barcode-matched sequence upstream of a fluorescent reporter gene. CRISPR activation of the fluorescent reporter will only occur in cells producing the matched sgRNA barcode, allowing precise identification and isolation of lineages of interest from heterogeneous populations.
Rabbit Anti BSA | |||
EnoGene | |||
Rabbit anti BNP | |||
MyBiosource | |||
Rabbit anti BNP | |||
MyBiosource | |||
Rabbit anti BNP | |||
MyBiosource | |||
Rabbit anti BNP | |||
MyBiosource |
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Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant
The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years www.joplink.net/tag-recombinants, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes.
This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55 mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30 °C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification.
Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.
MARK Antibody | |||
AAT Bioquest | |||
MARK antibody | |||
Fitzgerald | |||
MARK antibody | |||
Fitzgerald | |||
MARK Antibody | |||
SAB | |||
MARK Antibody | |||
SAB |
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Introduction of a Hexalysine (6 K) Tag Can Protect from N-Terminal Cleavage and Increase Yield of Recombinant Proteins Expressed in the Periplasm of E. coli
Recombinant expression of proteins in the periplasm of E. coli is frequently used for proteins containing disulfide bonds that are essential for protein folding and activity, as the cytosol of E. coli constitutes a reducing environment. The periplasm in contrast is an oxidative environment which supports proper protein folding. However, yields can be limited compared with cytoplasmic expression, and protocols must be adjusted to avoid overloading the periplasmic transportation machinery.
Another less-appreciated issue with periplasmic expression is the potential generation of unwanted N-terminal cleavage products, a persistent issue which we encountered when expressing the disulfide bond containing extracellular regions of several Helicobacter pylori adhesins (BabA, BabB, BabC, and LabA) in the periplasm of E. coli XL10 GOLD, a strain traditionally not used for proteins expression.
Here, we describe how introducing a C-terminal hexa-lysine (6 K) tag enhanced solubility and protected BabA from N-terminal proteolytic degradation (BabA), enabling crystallization and subsequent X-ray structural analysis. However. the same strategy had no advantageous effect for LabA, which using this protocol could be retrieved from the periplasm in relatively high yields (20-40 mg/L).
Recombinant Humanp21 Recombinant Protein | |||
ProSci | |||
TWEAK, recombinant / TNFSF12, recombinant (Human) | |||
PHOENIX PEPTIDE | |||
TAGLN Recombinant Protein (Rat) (Recombinant- Tag) | |||
ABM | |||
TAGLN2 Recombinant Protein (Rat) (Recombinant Tag) | |||
ABM | |||
TAGLN3 Recombinant Protein (Rat) (Recombinant Tag) | |||
ABM |
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Eimeria maxima Rhomboid-like Protein 5 Provided Partial Protection against Homologous Challenge in Forms of Recombinant Protein and DNA Plasmid in Chickens
Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX-EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX-EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX-EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays.
Recombinant Humanp21 Recombinant Protein | |||
92-035 | |||
TWEAK, recombinant / TNFSF12, recombinant (Human) | |||
054-85 | |||
TAGLN Recombinant Protein (Rat) (Recombinant- Tag) | |||
RP232205 | |||
TAGLN2 Recombinant Protein (Rat) (Recombinant Tag) | |||
RP232208 | |||
TAGLN3 Recombinant Protein (Rat) (Recombinant Tag) | |||
RP232211 |
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The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX-EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately.
After vaccination, pVAX-EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX-EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection.
The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis.
anti- Antibody^Polyclonal antibody control antibody | |||
LSMab09882 | |||
Lck antibody Antibody | |||
GWB-250026 | |||
H2B Antibody Antibody | |||
E11-184659 | |||
H2B Antibody Antibody | |||
MBS8529199-01mg | |||
H2B Antibody Antibody | |||
MBS8529199-01mLAF405L |
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A Novel Approach for the Production of Aggregation-Prone Proteins Using the Spidroin-Derived NT* Tag
Spiders have evolved proteins that can be kept in a highly concentrated soluble form in the silk gland yet rapidly assemble into stable silk fibers under certain environmental conditions. The transition between soluble and fibrillar states is partly regulated by the pH-sensitive N-terminal (NT) domain which has emerged as nature’s own solubility-enhancing domain. NT has an inherent capacity to keep the silk proteins’ partly hydrophobic and very aggregation-prone regions from premature fibrillation in spite of storage at enormous concentrations.
The genetically engineered double-mutant NT* shows increased solubility and stability and has arisen as a powerful tool for the production of aggregation-prone as well as other recombinant proteins. Here we describe a robust and highly efficient protocol for improved soluble expression of peptides and proteins by fusion to the NT* tag.
Ferret IL-6 Recombinant Mammalian N-Terminal 10xHis Tagged |
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each | 6349 EUR |
Ferret IL-6 Recombinant Mammalian N-Terminal 10xHis Tagged |
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each | 3865 EUR |
Ferret IL-6 Recombinant Baculovirus N-Terminal 10xHis Tagged |
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each | 9227 EUR |
Ferret IL-6 Recombinant Baculovirus N-Terminal 10xHis Tagged |
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each | 4458 EUR |
Ferret IL-6 Recombinant Baculovirus N-Terminal 10xHis Tagged |
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each | 5865 EUR |
Ferret IL-6 Recombinant Baculovirus N-Terminal 10xHis Tagged |
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each | 3070 EUR |
Ferret IL-6 Recombinant E Coli N-Terminal 10xHis Tagged |
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each | 4203 EUR |
Ferret IL-6 Recombinant E Coli N-Terminal 10xHis Tagged |
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each | 2494 EUR |
Ferret IL-6 Recombinant E Coli N-Terminal 10xHis Tagged |
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each | 2759 EUR |
Ferret IL-6 Recombinant E Coli N-Terminal 10xHis Tagged |
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each | 1837 EUR |
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Rat Cholesterol ELISA ELISA | |||
E01A11128 | |||
Rat Cholesterol ELISA ELISA | |||
E02C0745-48wellsplate | |||
Rat Cholesterol ELISA ELISA | |||
E02C0745-96wellsplate |
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