Protein Analysis and Purification

NanoBiT™ Assay: Transformational Technology for Studying Protein Interactions Named a Top 10 Innovation of 2015

Posted on by Michele Arduengo, PhD

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For three out of the last four years, we have been honored to have one of our key technologies named a Top 10 Innovation by The Scientist. This year the innovative NanoBiT™ Assay (NanoLuc® Binary Technology) received the recognition. NanoBiT™ is a structural complementation reporter based on NanoLuc® Luciferase, a small, bright luciferase derived from the deep sea shrimp Oplophorus gracilirostris.

Using plasmids that encode the NanoBiT complementation reporter, you can make fusion proteins to “report” on protein interactions that you are studying. One of the target proteins is fused to the 18kDa subunit; the other to the 11 amino acid subunit. The NanoBiT™ subunits are stable, exhibiting low self-affinity, but produce an ultra-bright signal upon association. So, if your target proteins interact, the two subunits are brought close enough to each other to associate and produce a luminescent signal. The strong signal and low background associated with a luminescent system, and the small size of the complementation reporter, all help the NanoBiT™ assay overcome the limitations associated with traditional methods for studying protein interactions.

The small size reduces the chances of steric interference with protein interactions. The ultra bright signal, means that even interactions among proteins present in very low amounts can be detected and quantified–without over-expressing large quantities of non-native fusion proteins and potentially disrupting the normal cellular environment. And the NanoBiT™ assay can be performed in real time, in live cells.

The NanoBiT™ assay is already being deployed in laboratories to help advance understanding of fundamental cell biology. You can see how one researcher is already taking full advantage of this innovative technology in the video embedded below:

Visit the Promega web site to see more examples more examples how the NanoBiT™ assay can break through the traditional limitations for studying protein interactions in cells.

You can read the Top 10 article in The Scientist here.

IdeZ Protease: A New Tool for the Characterization of Antibodies

Posted on by Promega
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Therapeutic monoclonal antibodies are large, complex molecules that undergo numerous post translational modifications (PTMs).  In-depth characterization of antibody PTMs remains a significant hurdle because their large size (~150 kDa) makes mass spectrometry analysis extremely challenging.

IdeS protease specifically cleaves IgGs into Fab and Fc fragments. This enzyme is highly specific and cleaves human IgG specifically at one site in the lower hinge region.  Because of the exquisite specificity of the enzyme, it produces highly homogeneous Fc and Fab fragments which are then readily analyzed using techniques such as mass spectrometry or HPLC.

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One of the drawbacks of IdeS is that it exhibits poor activity against mouse IgGs. IdeZ Protease is an immunoglobulin-degrading enzyme from Streptococcus equi subspecies zooepidemicus. It is an engineered recombinant protease overexpressed in E. coli. Like IdeS Protease, IdeZ Protease specifically cleaves IgG molecules below the hinge region to yield F(ab′)2 and Fc fragments.  Reduction of the digestion products produces three fragments of ~25kDa that are readily analyzed by LC-MS.

One of the key advantages of the IdeZ Protease is that it has significantly improved activity against mouse IgG2a and IgG3 subclasses compared to IdeS Protease. IdeZ Protease does not cleave mouse IgG1 or IgG2b.

Key technical parameters when digesting mouse IgGs utilizing IdeZ are the following:

• Add 1 unit of IdeZ Protease per 1µg of IgG to be digested.
• IdeZ Protease is most active in buffers at or near neutral pH. The recommended digestion buffer is 50mM sodium phosphate, 150mM NaCl (pH 6.6).
• Mouse IgG2a and IgG3 typically require 2–4 hours at 37°C  for complete digestion.
• IdeZ Protease has a histidine tag for easy removal if so desired.

Interrogating Protein Interactions: An Infographic for NanoBRET™ Assay Design

Posted on by Kelly Grooms

Yesterday my fellow blogger, Kari, posted a review of the ACS Chemical Biology paper describing a new BRET platform for analyzing protein-protein interactions. If you are interested in studying induction and inhibition of protein interactions in real time, take a look at the infographic below to learn how to develop a NanoBRET™ Assay to monitor your protein of interest.

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Purify and Conjugate Antibodies in a Single Workflow

Posted on by Promega

Isoform_Antibodies_LinkedInAntibodies labeled with small molecules such as fluorophore, biotin or drugs play a critical role in various areas of biological research,drug discovery and diagnostics. There are several limitations to current methods for labeling antibodies including the need for purified antibodies at high concentrations and multiple buffer exchange steps.

In a recent publication, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads.

Using a variety of fluorophores the researchers show that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry.

This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.

Literature Cited

Nidhi, N. et al. (2015) On-bead antibody-small molecule conjugation using high-capacity magnetic bead J. Immunol. Methods  http://dx.doi.org/10.1016/j.jim.2015.08.008

Cell-Free Expression Application: Antibody Screening

Posted on by Promega

TestPermissions2Ricin, derived from caster seeds, inhibits protein synthesis by binding to ribosomes, resulting in cell death. The protein is composed of two polypeptide chains: Ricin Toxin A chain and Ricin Toxin B chain. Ricin inhibits protein synthesis very quickly, and the cell or tissue damage begins within several hours. However, signs of poisoning often are not noted before significant damage has been done, making treatment difficult. Therapeutics that either block the ribosome binding site or compete with the toxin for binding are highly desired. Both antibodies and competitive ligands inhibited binding of the toxin to cell membranes.

A recent publication by Dong et al. (1), described a study to investigate the therapeutic effect of mAb 4C13, a monoclonal antibody against ricin. One of first experiments performed was to determine the general effect the inhibition of protein synthesis induced by ricin using cell-free expression.

In the study, the authors used T3 Coupled Reticulocyte Lysate Systems from Promega. Both ricin and mAb were diluted with saline. Aliquots of ricin (80 ng/ml) were mixed with an equal volume mAbs (1.6μg/ml) or saline alone and incubated at 4 °C for 1.5 h. A total volume of 4μl of sample was added into the reaction system (i.e, T3 Coupled Reticulocyte and plasmid DNA containing the lucifersase gene downstream of T3 RNA phage promoter). After incubation at 30 °C for 1.5 h, the products were cooled at −20 °C for 10 min. A total of 5μl of each reactive product containing synthesized luciferase was mixed with 50μl luciferase assay reagent pre-equilibrated to room temperature, and the fluorescence absorbance was measured immediately with the micro-ELISA Reader.

Positive results obtained from this preliminary experiment, led to more thorough experiments to determine the dosage effect using in vivo models (i.e., cell lines and mice) to characterize the cytotoxicity and binding activity of mAb 4C13. The mAB 4C13 was shown to be a effective in the mouse model.

Dong, N. et al. (2015) Monoclonal antibody , mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine 33, 3836–42

Sample preparation: A critical step for consistent protein phosphorylation data

Posted on by Promega

MSextractcroppedProtein phosphorylation is a very important protein post-translational modification that controls many cellular processes including metabolism, transcriptional and translation regulation, degradation of proteins, cellular signaling and communication, proliferation, differentiation, and cell survival (1). Approximately 35% of human proteins are phosphorylated. Phosphoproteins are low in abundance, and, therefore, are challenging to detect and characterize by mass spectrometry. Different enrichment systems have been developed to isolate phosphopeptides. Among these techniques, immobilized metal affinity chromatography (IMAC) using Fe3+ and Ga3+ has been widely used for the enrichment of phosphopeptides.

Typical experimental workflows are tedious and consist of numerous steps, including sample collection and cell lysis. One of the major challenges of the process is to maintain the in vivo phosphorylation state of the proteins throughout the preparation process

To evaluate the effect of sample collection protocols on the global phosphorylation status of the cell, a recent paper by Kashin et al. compared different sample workflows by metabolic labeling and quantitative mass spectrometry on Saccharomyces cerevisiae cell cultures (2).

Three different sample collection workflows were evaluated: two that used denaturating conditions and involved mixing of cell cultures with an excess of either ethanol (EtOH) at −80 °C or trichloroacetic acid (TCA), and a third under nondenaturing conditions and washing the cells in PBS.

Their data suggest that either TCA or EtOH sample collection protocols introduced lower collection bias than the PBS protocol. It was also suggested that similar studies be carried out to determine what effects sample preparation has on other post translation modifications such as acetylation or ubiquitination.

Literature Cited

  1. Thingholm T.E. et al, (2009) Analytical strategies for phosphoproteomics. Proteomics 9,1451–68
  2. Kanshin, E. et al. (2015)  Sample Collection Method Bias Effects in Quantitative Phosphoproteomics. J  Proteome Res. 14, 2998-04.

pH Reactive Dyes for Screening Antibody Internalization

Posted on by Promega
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Antibody drug conjugates (ADCs) are a new class of therapeutic drugs that uses antibodies to deliver highly toxic drug molecules specifically to the cancer cells. A key requirement for ADCs is the ability of antibody to bind to the cancer cells followed by internalization and subsequent release of drug inside the cells leading to cell apoptosis.

Traditionally, selection of lead antibody candidates for ADCs was done in a sequential workflow where antibodies were first selected based on their affinity followed by characterization involving antibody internalization and drug conjugation. However, there is evidence that high affinity doesn’t always correlate with good internalization and hence there is a need to screen antibodies for internalization properties in addition to their affinities.

Promega has developed a method that allows antibody to be screened for their internalization properties in a simple, plate-based format. The method uses pH sensor dyes (pHAb dyes), which are not fluorescent at neutral pH but become highly fluorescent at acidic pH. When antibody conjugated with pHAb dye binds to its antigen on the cancer cell membrane they are not fluorescent but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dye becomes fluorescent.

Fluorescence signal, for pHAb dyes conjugated using either amine or thiol chemistry, is minimal at pH>7 and increase significantly as the pH drops to pH 5.0, which is a typical pH in cell endosomal compartment. Moreover, pH response of free pHAb dye is similar to that of conjugated dye indicating that conjugation chemistry doesn’t influence the pH response of the dye.

Due to the high signal-to-background ratios of the dyes, plate-based internalization assays can be performed, enabling screening of large libraries of antibodies for their internalization properties, hopefully leading to improved identification of lead candidates for ADC applications.

Filter-Aided Sample Preparation before Mass Spec Analysis: An Evaluation of FASP and eFASP

Posted on by Promega

12271ma_800pxFilter-aided sample preparation (FASP) method is used for the on-filter digestion of proteins prior to mass-spectrometry-based analyses (1,2). FASP was designed for the removal of detergents, and chaotropes that were used for sample preparation. In addition, FASP removes components such as salts, nucleic acids and lipids. Akylation of reduced cysteine residues is also carried out on filter, after which protein is proteolyzed by use of trypsin on filter in the optimal buffer of the enzyme. Subsequent elution and desalting of the peptide-rich solution then provides a sample ready for LC–MS/MS analysis.

Erde et al. (3) described an enhanced FASP (eFASP) workflow that included 0.2% DCA in the exchange, alkylation, and digestion buffers,thus enhancing trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. DCA has been reported (4) to improve the efficiency of the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides.

In a recent publication (5) traditional FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). The results indicate that at the protein level, both methods extracted essentially the same number of hydrophobic transmembrane containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes.

The LC–MS/MS results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

  1. Manza, L. L. et al. (2005) Sample preparation and digestion for proteomic analyses using spin filters Proteomics  5, 1742–74.
  2. Wiśniewski, J. R. et al. (2009) Universal sample preparation method for proteome analysis Nat. Methods 6, 359–62.
  3. Erde, J. et al. (2014) Enhanced FASP (eFASP) to increase proteomic coverage and sample recovery for quantitative proteome experiments. J. Proteome Res. 13, 1885–95.
  4. Lin, Y. et al. (2008) Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins Anal. Biochem.  377, 259–66.
  5. Nel. A. et al. (2015) Comparative Reevaluation of FASP and Enhanced FASP methods by LC-MS/MS/ J Proteome Res. 14, 1637–42.

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Posted on by Michele Arduengo, PhD
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) .

Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

HaloTag® Research Application: Detection of Cancer Biomarkers

Posted on by Promega

10242TAAntibodies labelled with radioisotopes or the sequential administrationof an antibody and a radioactive secondary agent facilitate the in vivo detection and/or characterisation of cancers by positron emission tomography (PET) or by single-photon emission computed tomography (SPECT) imaging.

There are drawbacks to both methods, including prolonged exposure to radiation and  ensuring that both the antibody and the radiolabelled secondary agent are suitably designed so that they bind rapidly upon contact at the tumor.

A recent publication (1) investigated a alternative method utilizing the HaloTag® dehalogenase enzyme HaloTag® is a dehalogenase enzyme (33 kDa) that contains an engineered cavity designed to accommodate the reactive chloroalkane group of a HaloTag® ligand (HTL). Upon entering the enzyme cavity, the terminal chlorine atom rapidly undergoes nucleophilic displacement, and a covalent adduct is formed, effectively anchoring the HaloTag® ligand in a precise location.

Three new HaloTag® ligands were synthesized and each labelled with the SPECT radionuclide indium-111  111In-HTL-1  and the dual-modality HaloTag® ligands,111In-HTL-2 and111;In-HTL-3 containing TMR which allows complementary imaging data).

For the validation of the pretargeting strategy based on these HaloTag® ligands, the target human epidermal growth factor receptor 2 (HER2)was selected. Trastuzumab (Herceptin®) was selected as the primary targeting agent and was modified with HaloTag® protein via the trans-cyclooctene/tetrazine ligation.

All three 111In-labelled HaloTa®g ligands exhibited significantly higher binding to the HER2 expressing when compared to negative controls.

Literature Cited

Knight, J. C et al.(2015) Development of an enzymatic pretargeting strategy for dual-modality imaging. Chem. Commun. 51, 4055–8.