Tags For The Purification Of Recombinant Proteins

In the process of expression and obtaining of fusion proteins , one of the most critical and most challenging stages is the purification of recombinant proteins . This step is a special challenge when working with uncharacterized proteins, or with proteins for which there is no good antibody.

The tags affinity usually generally small amino acid sequences whose fusion to the target protein by genetic engineering techniques allows, in theory, the purification of any protein by affinity chromatography, without having prior knowledge of the biochemical properties Of the same.

In this post we bring you a compilation of the most used affinity tags for the purification of recombinant proteins.

The choice of the fusion tag that we want to add to our target protein will depend mainly on the purification priorities, the size of the tag and the cost of the chromatography resins.

Although the purification of recombinant proteins by affinity chromatography against fusion tags is generally used as a single step, in cases where higher purity is required this technique can be used as a preliminary step within a more complex purification procedure, or simultaneously fusing two tags to the 3 ‘and 5’ ends of the target protein, and carrying out a double affinity purification against each of them.

Below we summarize the characteristics of the affinity tags most used in the purification of recombinant proteins:

1.- HIS TAG

  • Name : Polyhistidine
  • Size : 6 aa
  • Features :
  • It is the most commonly used tag.
  • Due to its small size, it rarely interferes with protein function.
  • Likewise, the elution by means of the imidazole gradient is not very aggressive, which allows preserving the immunogenicity of the proteins.
  • The purification of recombinant histidine tag proteins can be performed by IMAC (Immobilized Metal Affinity Chromatography), generally with Ni2 + columns, or by primary anti-polyhistidine antibodies.
  • In the case of the proteins expressed in E. coli, purification by means of the histidine tag can reach a purity of 80%. However, in other systems such as insect or mammalian cells, the purity achieved may be lower due to the high percentage of histidine residues that the proteins present in these systems.

2.- GST TAG

  • Name : Glutathione S-transferase
  • Size : 26 kDa
  • Features :
  • Another of the most popular tags and one of the largest.
  • It has the ability to increase the expression and solubility levels of the fusion protein.
  • Due to its large size, it could interfere with the function of the protein.
  • Glutathione elution is not very aggressive, which allows the functionality and antigenicity of the fusion protein to be preserved in most cases.
  • The purification of recombinant GST tag proteins can be carried out using glutathione resins, and using anti-GST antibodies.
  • The GST tag can be separated and removed from the fusion protein, while still attached to the resin.
  • GST-bound proteins are generally expressed at high levels in E. coli, which can lead to the formation of aggregates in inclusion bodies.

3.- MBP TAG

  • Name : Maltose-binding protein.
  • Size : 45kDa
  • Features :
  • It is the largest tag.
  • It has the ability to increase the levels of expression and solubility of the fusion protein, also contributing to its correct folding.
  • Due to its large size, it could interfere with the function of the protein.
  • Purification of recombinant MBP tag proteins can be purified using amylose resins and anti-MBP antibodies.
  • The MBP tag cannot be separated and removed from the fusion protein while it is still bound to the resin, and the protein must be eluted prior to cleavage.
  • As with the GST tag, MBP-bound proteins are generally expressed at high levels in E. coli, which can lead to the formation of aggregates in inclusion bodies.

4.- CBP TAG

  • Name : Calmodulin-binding peptide
  • Size : 26aa / 4kDa
  • Features :
  • Being a small tag, the probability that it affects the properties of the protein of interest is very low.
  • One of the main advantages of this system lies in the mild binding and elution conditions, which contribute to the fusion protein maintaining its native form after purification.

5.- STREPTAVIDIN / BIOTIN BASED TAGS

  • Name : BCCP (biotinylation signal peptide) + biotin
  • Size : 100aa
  • Features :
  • Proteins that include the BCCP tag can be biotinylated by the enzyme biotin ligase.
  • Once biotinylated, the purification of recombinant proteins can be carried out by affinity chromatography with avidin resin.
  • While nonspecific binding to avidin in E. coli is very low, in other systems such as mammalian cells, proteins other than the target protein could be eluted simultaneously.

6.- EPITOPE TAGS

  • Name : FLAG, Hemagglutinin (HA), c-myc, T7, etc.
  • Size : 8 aa (FLAG), 31aa (HA), 11aa (c-myc), 11aa (T7)
  • Features :
  • Epitope tags are usually small sequences that act at the level of the epitopes of the protein of interest (antibody recognition sites).
  • The purification of recombinant proteins with epitope tags can be performed by affinity chromatography, immobilizing specific primary antibodies.
  • It is not the most recommended system in the case of highly insoluble proteins or those with a tendency to form aggregates.

In conclusion , the choice of the tag (and the cleavage method, if applicable) for the purification of recombinant proteins , should be made taking into account the particularities of our protein of interest. For proteins that are well expressed, the use of some of the simplest affinity tags such as polyhistidine tail would suffice, while in the case of those proteins that present greater expression difficulties, it would be necessary to resort to tags that also facilitate solubility and folding such as GST or MBP.

On the other hand, it should also be borne in mind that small tags hardly interfere with the structure, activity and characteristics of the target protein, while larger tags (GST, MBP) can have a significant impact both in the structure and in the biological activity of the fusion protein, and it would be necessary to assess in each case the desirability of cleaving said tag once the protein of interest has been purified.

5 Reasons To Use Chicken Antibodies

The chicken antibodies , known as IgY or avian antibodies, first isolated from the yolk of the s end. XIX. Although IgY as a tool for biomedical research is not the most widespread option today, there is no doubt that it is gaining more ground among the scientific community. And this is due to the considerable advantages they provide in certain cases compared to polyclonal antibodies traditionally produced in mammals.

In this post we collect the top 5 reasons to use chicken antibodies against polyclonal antibodies (IgG) produced in rabbits, mice, goats, or other mammals.

1.- Phylogenetic difference between chickens and humans

Between humans and chickens there are 310 million years of evolution, which makes the difference between homologous proteins of these two species greater than that between human proteins with respect to their counterparts in other mammals.

This difference translates into a greater number of epitopes recognizable as “foreign” by the host, causing greater immunogenicity and specificity of the immune response.

In short, chicken antibodies (IgY) represent a clear advantage over polyclonal antibodies obtained in mammals in those cases in which the target protein is a highly conserved protein between species.

2.- Structural differences between IgY and IgG

Chicken antibodies show several structural differences from IgG, such as the presence of an additional constant domain in the heavy chain.

But without a doubt, the most significant difference is that chicken antibodies lack constant domain (Fc), with its consequent advantages:

  • They do not activate the complement system.
  • They do not interact with Fc receptors.
  • They do not interact with endogenous mammalian anti-Fc antibodies.

3.- Stability

Chicken antibodies (IgY) have a greater stability against IgG, which allows them to be stored under refrigerated conditions (+ 4ºC) for longer periods of time (up to years versus days or months).

4.- Animal welfare

Although chicken antibodies (IgY) are also logically present in the blood of animals, they can also be isolated from egg yolks laid by immunized chickens.

This fact avoids the invasive and traumatic process that constitutes obtaining polyclonal antibodies by bleeding the animals, as well as their death after exsanguination.

5.- Low cost and higher performance

Maintaining chickens and chickens as research animals is more economical than other animals such as rabbits, goats, etc.

At the same time, the production of specific antibodies in chickens is much higher, obtaining a higher yield than in the case of mammals (up to 10-20 times more than the production of polyclonal antibodies in rabbit).

And to all this is added the shorter production time, since in the case of chicken antibodies specific IgY can be obtained in just 25 days.

In summary, the production of chicken antibodies involves:

  • Lower cost.
  • Higher performance.
  • Less production time.
A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) current an unprecedented alternative to advance human well being by providing another and renewable cell useful resource for mobile therapeutics and regenerative medication.

The current demand for prime quality hPSCs to be used in each analysis and medical research underscores the must develop applied sciences that may simplify the cultivation course of and management variability.

Here we describe the improvement of a sturdy, outlined and xeno-free hPSC medium that helps dependable propagation of hPSCs and technology of human induced pluripotent stem cells (hiPSCs) from a number of somatic cell varieties; long-term serial subculturing of hPSCs with every-other-day (EOD) medium alternative; and banking absolutely characterised hPSCs.

The hPSCs cultured in this medium for over 40 passages are genetically secure, retain excessive expression ranges of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm.

Importantly, the medium performs an integral position in establishing a cGMP-compliant course of for the manufacturing of hiPSCs that can be utilized for technology of clinically related cell varieties for cell alternative remedy purposes.

A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.
A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

Silver nanoparticles inhibit fish gill cell proliferation in protein-free tradition medium.

While short-term exposures of vertebrate cells, corresponding to from fish, could be carried out in outlined, serum-free media, long-term cultures typically require addition of development components and proteins, usually equipped with a serum complement. However, proteins are recognized to change nanoparticle properties by binding to nanoparticles.

Therefore, in order to have the ability to research nanoparticle-cell interactions for prolonged durations, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was tailored to proliferate in a business, serum-free medium, InVitrus VP-6. The newly tailored cell pressure was named RTgill-W1-pf (protein free). These cells proliferate at a velocity much like the RTgill-W1 cells cultured in a completely supplemented medium containing 5% fetal bovine serum.

As nicely, they have been efficiently cryopreserved in liquid nitrogen and absolutely recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium can’t absolutely assist long-term tradition of the RTgill-W1 pressure.

The RTgill-W1-pf cell line was subsequently utilized to research the impact of silver nanoparticles (AgNP) on cell proliferation over a interval of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This impact correlated with excessive ranges of silver being related to the cells.

The new cell line, RTgill-W1-pf, can function a singular illustration of the gill cell-environment interface, providing novel alternatives to review nanoparticle-cell interactions with out serum protein interference.

Cell-free culture conditioned medium elicits pancreatic β cell lineage-specific epigenetic reprogramming in mice.

Cell-free culture conditioned medium elicits pancreatic β cell lineage-specific epigenetic reprogramming in mice.

There are a number of obstacles to beat previous to reaching mobile reprogramming of pancreatic β cells in vitro and in vivo.

The current research demonstrated that the switch of epigenetic phenotypes was achieved in the cell-free conditioned medium (CM) of pancreatic insulinoma MIN6 cell cultures.

The comparability of a subpopulation of MIN6, m14 and m9 cells indicated that MIN6-m14 cells had been extra liable to mobile reprogramming. Epigenetic profiling revealed that the transcription issue pancreas/duodenum homeobox protein 1 (Pdx1) was differentially related among the many clones.

The culture of differentiated adipocytes in the CM of MIN6-m14 cells resulted in the induction of insulin mRNA expression, and was accompanied by epigenetic occasions of Pdx1 binding.

The epigenetic profiling indicated that Pdx1 is preferentially related to a beforehand uncharacterized area of the endoplasmic reticulum (ER) disulfide oxidase, ER oxidoreductin 1 gene.

Therefore, the outcomes of the current research indicated that the CM of MIN6 cells was capable of induce a pancreatic β cell-like phenotype in differentiated adipocytes.

These information present further assist for the utility of cell-free CM for mobile reprogramming.

Cell-free culture conditioned medium elicits pancreatic β cell lineage-specific epigenetic reprogramming in mice.
Cell-free culture conditioned medium elicits pancreatic β cell lineage-specific epigenetic reprogramming in mice.

A modified methodology by differential adhesion and serum-free culture medium for enrichment of most cancers stem cells.

UNASSIGNEDIn this research, we confirmed a modified methodology for the isolation of most cancers stem cells (CSCs) utilizing a mix of differential adhesion methodology and serum-free culture medium (SFM) methodology.

UNASSIGNEDTrypsin-sensitive cells and trypsin-resistant cells had been remoted from MB49, EJ, and SK-OV-Three cells utilizing a mix of differential adhesion methodology and SFM methodology.

The CSCs markers expression of trypsin-resistant cells was verified by the circulate cytometry, the Western blotting, and the quantitative polymerase chain response. Functional comparisons had been verified by the resistance to chemotherapy assay, the transwell assay, and the tumor xenograft formation assay.

UNASSIGNEDTrypsin-resistant cells had been remoted efficiently. They had been recognized with excessive expression of CSCs markers and possessed increased resistance to chemotherapy, larger migration in vitro and stronger tumorigenic skills in vivo.

UNASSIGNEDTrypsin-resistant cells confirmed particular CSCs characterizations. They had been capable of be remoted efficiently with a modified methodology by a mix of differential adhesion methodology and SFM methodology.

Optimizing a serum-free/xeno-free culture medium for culturing and promoting the proliferation of human dental pulp stem cells.

Optimizing a serum-free/xeno-free culture medium for culturing and promoting the proliferation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) maintain nice promise for utilization in tissue restore and regenerative drugs.

Routinely, culture media used for culturing stem cells are supplemented with animal serum for promoting progress and profitable upkeep of stem cells.

However, there may be a rising demand for optimizing a well-defined culture media that might safely improve the efficacy and reproducibility of the cultured cells. In this research, we aimed toward optimizing a serum-free/xeno-free culture medium.

A cocktail of numerous dietary supplements supposed to counterpoint DPSCs proliferation in outlined concentrations was designed.

It consisted of recombinant human fundamental fibroblast progress issue (hbFGF), insulin transferrin selenium (ITS), ascorbic acid (vitamin C), Beta mercaptoethanol and ldl cholesterol.

The impact of this optimized media on the proliferation of DPSCs was assessed by MTT assay and circulate cytometric evaluation (FACS) of early apoptotic marker annexin V. Expression of stemness-related genes (OCT4, SOX and NANOG) was assessed by qRT-PCR.Proliferation outcomes by MTT illustrated a important improve in the proliferation fee of DPSCs cultured in the proposed media.

FACS evaluation of annexin V expression was nil. Expression of OCT4, SOX and NANOG genes was additionally up-regulated.

The proposed mixture of dietary supplements utilized in the proposed culture media efficiently elevated the proliferation potential of DPSCs along with enhancing the stemness properties.

Thus, it may be thought of a promising and secure substitute to conventional animal derived dietary supplements like fetal bovine serum (FBS).

Optimizing a serum-free/xeno-free culture medium for culturing and promoting the proliferation of human dental pulp stem cells.
Optimizing a serum-free/xeno-free culture medium for culturing and promoting the proliferation of human dental pulp stem cells.

[Retracted] Serum‑free‑medium‑kind mesenchymal stem cell culture supernatant exerts a protecting impact on A549 lung epithelial cells in acute lung damage induced by H2O2.

The authors want to retract their analysis article entitled ‘Serum‑free‑medium‑kind mesenchymal stem cell culture supernatant exerts a protecting impact on A549 lung epithelial cells in acute lung damage induced by H2O2’, revealed in Oncology Reports 40, 3033‑3039, 2018.

After the publication of this text, the authors have develop into involved that there have been flaws of their research design which have known as into query the reported outcomes.

On repeating sure of the experiments, the authors discovered that the Nrf2‑Keap1‑ARE signaling pathway solely has a function in the lung epithelial cell damage mannequin, whereas it doesn’t serve a function in the A549 mannequin.

Further research are required to validate the function of the Nrf2‑Keap1‑ARE signaling pathway and the apoptosis‑related proteins. In explicit, the outcomes introduced in Fig. 5, displaying the distinction between Bax and Bcl‑2, look like incorrect. For these causes, the authors have determined to retract the article from the publication.

All the named authors on the paper comply with this retraction. The authors sincerely apologize for any inconvenience that may consequence from the retraction of this text. [the original article was published in the Oncology Reports 40: 3033‑3039, 2018; DOI: 10.3892/or.2018.6656].