Protein Analysis and Purification

His-Tagged Fusion Proteins: Application Update

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Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;
Crystal structure of Polyhistidine tagged recombinant catalytic subunit of cAMP-dependent protein kinase. Credit: StructureID=1fmo; DOI=http://dx.doi.org/10.2210/pdb1fmo/pdb;

Researchers often need to purify a single protein for further study. One method for isolating a specific protein is the use of affinity tags. Affinity purification tags can be fused to any recombinant protein of interest, allowing fast and easy purification following a procedure that is based on the affinity properties of the tag.

The most commonly used tag to purify and detect recombinant expressed proteins is the polyhistidine tag. Protein purification using polyhistidine tags relies on the affinity of histidine residues for immobilized metal such as nickel, which allows selective protein purification. The metal is immobilized to a support through complex formation with a chelate that is covalently attached to the support.

Polyhistidine tags offer several advantages for protein purification. The small size of the polyhistidine tag renders it less immunogenic than other larger tags. Therefore, the tag usually does not need to be removed for downstream applications following purification.

A large number of commercial expression vectors that contain polyhistidine are available. The polyhistidine tag may be placed on either the N- or C-terminus of the protein of interest.

And finally, the interaction of the polyhistidine tag with the metal does not depend on the tertiary structure of the tag, making it possible to purify otherwise insoluble proteins using denaturing conditions. The resulting purified protein can be used for a variety of applications.

The following references illustrate examples of some of the most common post purification applications with fusion proteins containing a polyhistidine tag:

Enzymatic assays

  1. Negi, V-S. et al. (2014) A carbon nitrogen Lyase from Leucaena leucocephala catalyzes the first Step of mimosine degradation. Plant Physiol. 164,  922–34.
  2. Yu, S. et al. (2013) Syk Inhibits the activity of protein kinase A by phosphorylation tyrosine of the catalytic subunit. J. Biol. Chem. 288, 10870-81.
  3. Rusconi, B. et al. (2013) Discovery of catalases in members of the Chiamydiales order. J. Bact. 195, 3543–51.

Structural analysis

  1. Araiso, Y. et al. (2014) Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB a novel subunit assembly in tRNA –dependent amidotransferases. Nucl.Acids. Res.(available only online).
  2. Someya, T. et al. (2012) Crystal Structure of Hfq from Bacillus subtilis in complex with SELEX-dervived RNA aptamer: insight into RNA-binding properties of bacterial Hfq. Nucl. Acid Res. 40, 1856-67.

Protein pulldowns

  1. Yun, S-C. et al. (2010) Pmr a Histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function. J.Bact. 192, 4720–31.
  2. Haim, H. et al. (2010)  Cytokeratin 8 interacts with clumping factor B: a new possible virulence factor target. Microbiology 156, 3710-21.

Additional Resources
His-tagged Protein Purification Systems

CheckMate™ Mammalian Two-Hybrid System: Application Update

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Assay principle for CheckMate™ Mammalian Two-Hybrid System.
Assay principle.

In the CheckMate™ Mammalian Two-Hybrid System, the pBIND Vector contains the yeast GAL4 DNA-binding domain upstream of a multiple cloning region, and the pACT Vector contains the herpes simplex virus VP16 activation domain upstream of a multiple cloning region. The two genes encoding the two potentially interactive proteins of interest are cloned into pBIND and pACT Vectors to generate fusion proteins with the DNA-binding domain of GAL4 and the activation domain of VP16, respectively. The pG5luc Vector contains five GAL4 binding sites upstream of a minimal TATA box, which in turn, is upstream of the firefly luciferase gene (luc+). The pGAL4 and pVP16 fusion constructs are transfected along with pG5luc Vector into mammalian cells. Interaction between the two test proteins, as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression over the negative controls. Traditionally mammalian two hybrid analysis was used to confirm initial data obtained from yeast two hybrid experiments.

Due to enhanced bioinformatics information and the development of improved co-immunoprecipiation/pulldown procedures/technology, there is a growing trend to use only mammalian cells to characterize protein:protein interactions. The following references illustrate the use of the CheckMate™ system to complement  other techniques to characterize protein;protein interactions using only mammalian cells .

Bagchi, P. et al. (2013) Molecular Mechanism behind Rotavirus Nsp-1 Mediated PI3 Kinase Activation: Interaction between NSP1 and the p85Subunit of PI3 kinase. J. Vir. 87, 2358-62.

Greninger, A. et al. (2013) ACBD3 Interaction with TBC1 domain 22 protein is differentially affected by Enteroviral and Kobuviral 3A protein binding.  mBio 4, 00098-13.

Patki, M. et al. ( 2013) The ETS Domain Transcription Factor ELK1 Direct a critical component of growth signalling by Androgen Receptor in prostrate cancer cells. J.Biol. Chem. 288 11047-65

Konig, H-G. (2012) Fibroblast growth factor homologous factor 1 interact with NEMO to regulate NF-kappaB signaling in neurons J. Cell Sci. 125, 6058-70

Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments

Posted on by Ben Schmidt

In vitro translation of proteins through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function.  If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale).  There are many systems out there, and knowing which to use can sometimes be difficult.  Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA.  But when you want to make your own RNA templates to add to lysates, then there are additional concerns.

artists concept of in vitro translation
A protein chain being produced from a ribosome.

Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason.  Extracting it requires the utmost care in technique and elimination of nucleases.  Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease).  Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.

The first step for using cell-free expression systems with RNA templates is to make the RNA.  Here are some tips that will ensure success.

Continue reading “Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments”

Asp-N Protease: Applications Update

Posted on by Promega
TrypsinLysC Page 2

Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for  in-solution or in-gel digestion reactions.

The following references illustrate the use of Asp-N in recent publications:

Protein sequence coverage

  1. Jakobsson, M et al. (2013)  Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
  2. Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I.  J. Biol. Chem. 288, 24799–08.

Glycoprotein analysis

  1. Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
  2. Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
  3. Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”.  J. Biol. Chem. 288, 30192–09.

Phosphoprotein analysis

  1. Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.

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Cell-free Expression: A System for Every Need

Posted on by Kelly Grooms

6634MA

Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in traditional E. coli expression systems. Continue reading “Cell-free Expression: A System for Every Need”

Novel Application for ProteaseMAX Surfactant: Cell Lysis

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ProteaseMax Surfactant


The novel mass spectrometry compatible surfactant sulfonate-(sodium 3-((1-(furan-2-yl)undecyloxy) carbonylamino)-propane-1-sulfonate (i.e.ProteaseMAX) facilitates both in-gel and in-solution digestion applications by reducing the time required, enabling protein solubilization/denaturation and increasing peptide/protein identifications.

A new application was highlighted in a recent publication (1) which utilized ProteaseMAX to lyse cells prior to trypsin digestion and subsequent mass spec analysis. The composition of the buffer determines the overall efficiency of cell lysis, dissociation of protein complexes, protein solubility and ease of removal prior to LC/MS-MS analysis.

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When compared to lysis buffers containing either urea or SDC, ProteaseMAX provided the optimal number of identified peptides/proteins.
In addition it can be easily removed from the lysate by acidic precipitation.

Reference

  1. Pirmoradian, M. et al. (2013). Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol. Cell. Prot. 12, 3330–8.

ProTEV Protease Compound Compatibility Analysis

Posted on by Promega
Cleavage of 20µg of GST-MBP fusion protein with ProTEV protease after 60 minutes at 30°C.
Cleavage of 20µg of GST-MBP fusion protein with ProTEV protease after 60 minutes at 30°C.

Many proteins are expressed as fusion partners with affinity tags, such as the HaloTag® fusion, glutathione-S-transferase (GST) or maltose binding protein (MBP), to selectively bind the proteins using affinity purification resins. While such resins yield high-purity protein quickly, the large affinity tags are undesirable for some downstream applications. Most expression vectors are designed with a specific protein cleavage site between the two fusion partners to remove the affinity tag after purification. ProTEV Protease recognizes a rare amino acid sequence, EXXYXQ, where X is any amino acid, and cleaves after the glutamine residue.

ProTEV Plus functions over a broad pH and temperature range. In a recent study the enzymatic activity of ProTEV Plus in the presence of various compounds (Table 1) commonly found in protein purification protocols were evaluated.

Continue reading “ProTEV Protease Compound Compatibility Analysis”

Trypsin/Lys-C Mix: Alternative for standard trypsin protein digestions

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Trypsin/Lys-C Mix, Mass Spec Grade, is a mixture of Trypsin Gold, Mass Spectrometry Grade, and rLys-C, Mass Spec Grade. The Trypsin/Lys-C Mix is designed to improve digestion of proteins or protein mixtures in solution.It is a little known fact that trypsin cleaves at lysine residues with lesser efficiency than at arginine residues. Inefficient proteolysis at lysine residues is the major cause of missed (undigested) cleavages in trypsin digests.

11788MA


Supplementing trypsin with Lys-C enables cleavage at lysines with excepetional efficiency and specificity. Following the conventional trypsin digestion protocol (i.e., overnight incubation at nondenaturing conditions, reduction,alkylation, 25:1 protein:protease ratio [w/w], mix and incubate overnight at 37°C.) Replacing trypsin with Trypsin/Lys-C Mix in this conventional protocol leads to multiple benefits for protein analysis including more accurate mass spectrometry-based protein quantitation and improved protein mass spectrometry analytical reproducibility.

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Site-specific copy number variations in cancer: A story begins to unfold

Posted on by Michele Arduengo, PhD

Designed by Nick Klein for ISO-form, courtesy of Promega.
Designed by Nick Klein for ISO-form, courtesy of Promega.

Tumor cells are characterized by many features: including uncontrolled proliferation, to loss of contact inhibition, acquired chromosomal instability and gene copy number changes among them. Some of those copy number changes are site-specific, but very little is known about the mechanisms or proteins involved in creating site-specific copy number changes. In a recently published Cell paper, Black and colleagues, propose a mechanism for site-specific copy number variations involving histone methylation proteins and replication complexes.

Previous work from Klang et al. had shown that local amplification of chromosomal regions occurs during S phase and that chromatin structure plays a critical role in this amplification (2), and other work by Black and colleagues (3) implicated KDM4A in changing timing of replication by altering chromatin accessibility in specific regions. Other research also had shown that KDM4A protein levels influence replication initiation and that KDM4A has a role in some DNA damage response pathways (4,5).  Looking at the body of work, Black et al. hypothesized that KDM4A, with its roles in replication, might possibly provide link into the mechanism of site-specific copy number variation in cancer. Continue reading “Site-specific copy number variations in cancer: A story begins to unfold”

Convenient, Non-Radioactive Detection of Isoaspartate

Posted on by Promega
Structure of the PCMT1 protein. Based on PyMOL rendering of PDB 1i1n. Licensed under creative commons http://creativecommons.org/licenses/by-sa/3.0/deed.en
Structure of the PCMT1 protein. Based on PyMOL rendering of PDB 1i1n. Licensed under creative commons http://creativecommons.org/licenses/by-sa/3.0/deed.en

The ISOQUANT® Isoaspartate Detection Kit is intended for quantitative detection of isoaspartic acid residues in proteins and peptides, which can result from the gradual, nonenzymatic deamidation of asparagine or rearrangement of aspartic acid residues.

The ISOQUANT® Kit is designed to provide information regarding the global formation of isoaspartic acid residues at Asn and Asp sites, not at each site separately.

The deamidation of asparagine residues and rearrangement of aspartic acid residues is characterized by the formation of a succinimide intermediate that resolves to form a mixture of isoaspartic acid (typically 70–85%) and aspartic acid.
The rate and extent of isoaspartic acid formation can vary widely among proteins, depending on the amino acid sequence and size of the target protein. Deamidation of Asn residues has been observed most frequently at Asn-Gly and Asn-Ser sites within proteins.

The ISOQUANT® Isoaspartate Detection Kit uses the enzyme Protein Isoaspartyl ethyltransferase (PIMT) to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to isoaspartic acid. Spontaneous decomposition of this methylated intermediate results in the release of methanol and reformation of the succinimide.

References:

Wang, W. et al. (2012) Quantification and characterization of antibody deamidation by peptide mapping with mass spectrometry. Int. J. Mass. Spec. 312, 107–13.

Grappin, P. et al. (2011) New proteomic developments to analyze protein isomerization and their biological significance in plants. J. Proteomics, 74, 1475–82.

Yang, H. and Zubarev, R.A. (2010) Mass spectrometric analysis of asparagine deamidation and aspartate isomerization in polypeptides. Electrophoresis 31, 1764–71.

Sinha, S. et al. (2009) Effect of protein structure on deamidation rate in the Fc fragment of an IgG1 monoclonal antibody. Protein Sci. 18, 1573–84.