20
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

      research-article

      , , *

      PLoS ONE

      Public Library of Science

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His 6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

          Related collections

          Most cited references 27

          • Record: found
          • Abstract: found
          • Article: not found

          Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

          Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Protein splicing and related forms of protein autoprocessing.

             H Paulus (1999)
            Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor.

              Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteins, and contains a few small peptides of hitherto unknown functions. We demonstrate that two of these peptides inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These 'silk proteinase inhibitors' 1 and 2 (SPI 1 and 2) are produced in the middle section of the silk-secreting glands prior to cocoon spinning and their production is controlled at transcription level. The full length cDNA of pre-SPI 1 contains 443 nucleotides and encodes a peptide of 76 amino-acid residues, of which 20 make up a signal sequence. The mature SPI 1 (6056.7 Da, 56 residues) is a typical thermostable Kunitz-type proteinase inhibitor with Arg in P1 position. The cDNA of pre-SPI 2 consists of 260 nucleotides and yields a putative secretory peptide of 58 amino-acid residues. The functional SPI 2 (3993 Da, 36 residues) is a single-domain Kazal-type proteinase inhibitor with unique structural features: free segment of the N-terminus is reduced to a single amino-acid residue, lack of CysI and CysV precludes formation of the A-ring and provides increased flexibility to the C-ring, and absence of several residues around the normal position of CysV shortens and changes the alpha helix segment of the protein. The structure reveals that the length and arrangement of the B-ring, including exposure of the P1 residue, and the position of the C-terminus relative to the B-loop, are essential for the activity of the Kazal-type inhibitors.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                4 May 2012
                : 7
                : 5
                Affiliations
                [1]Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
                University of Cambridge, United Kingdom
                Author notes

                Conceived and designed the experiments: EK ABC WB. Performed the experiments: EK ABC. Analyzed the data: EK ABC WB. Contributed reagents/materials/analysis tools: WB. Wrote the paper: EK ABC WB.

                Article
                PONE-D-12-03625
                10.1371/journal.pone.0036350
                3344860
                22574150
                84ced8d5-5c5c-4dca-8943-24303ffc7310
                Kopera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 9
                Categories
                Research Article
                Biology
                Biochemistry
                Proteins
                Protein Chemistry
                Biophysics
                Protein Chemistry
                Computational Biology
                Macromolecular Structure Analysis
                Protein Structure
                Synthetic Biology
                Chemistry
                Chemical Reactions
                Hydrolysis
                Inorganic Chemistry
                Bioinorganic Chemistry

                Uncategorized

                Comments

                Comment on this article