Simple and Efficient Purification of Recombinant Proteins Using the Heparin‐Binding Affinity Tag

Srinivas Jayanthi1, Ravi Kumar Gundampati1, Thallapuranam Krishnaswamy Suresh Kumar1

1 Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 6.16
DOI:  10.1002/cpps.41
Online Posting Date:  November, 2017
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Abstract

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin‐protein interactions. Based on the structural knowledge available from the FGF1‐heparin interaction studies, we have designed a novel heparin‐binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost‐effective purification of recombinant proteins of interest. HBP‐tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP‐fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP‐fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc.

Keywords: affinity chromatography; fusion tag; heparin sepharose; recombinant protein; resin

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cloning and Expression of Recombinant Heparin‐Binding Peptide (HBP) Fusion Proteins
  • Basic Protocol 2: Protein Purification Using Heparin Sepharose Affinity Chromatography
  • Alternate Protocol 1: Purification of HBP‐Fusion Proteins Under Denaturing Conditions
  • Support Protocol 1: ON‐ and OFF‐Column Cleavage of HBP Fusion Tag from the Target Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cloning and Expression of Recombinant Heparin‐Binding Peptide (HBP) Fusion Proteins

  Materials
  • Luria‐Bertani (LB) medium, pH 7.2
  • Antibiotic stock (ampicillin 100 mg/ml)
  • Frozen glycerol stock of bacterial cell culture containing the HBP vector with desired gene of interest
  • 1 M IPTG
  • 1× PBS
  • 2‐liter Erlenmeyer flasks
  • Temperature‐controlled environmental shaker
  • UV‐vis spectrophotometer
  • 500‐ml centrifugation bottles
  • 50‐ml conical tubes
  • Refrigerated centrifuge

Basic Protocol 2: Protein Purification Using Heparin Sepharose Affinity Chromatography

  Materials
  • Heparin Sepharose resin (GE Healthcare)
  • 20% (v/v) ethanol
  • Equilibration buffer (see recipe)
  • Frozen bacterial cell pellet containing the HBP‐fusion protein (from protocol 1)
  • Sodium phosphate buffer
  • Elution buffer (see recipe)
  • Protease‐inhibitor cocktail (Sigma)
  • 1.6 × 20‐cm Econo column (BioRad)
  • Econo UV monitor
  • Low‐flow peristaltic pump
  • Ultrasonicator
  • French cell press
  • Oakridge tubes
  • 50‐ml conical tubes (VWR Scientific Inc.)
  • Gradient mixer
  • High‐speed refrigerated centrifuge (Beckman Coulter)
  • Millipore centrifugal ultrafiltration devices, with appropriate molecular weight cutoffs.

Alternate Protocol 1: Purification of HBP‐Fusion Proteins Under Denaturing Conditions

  Materials (also see protocol 2)
  • Equilibration buffer containing 8 M urea.

Support Protocol 1: ON‐ and OFF‐Column Cleavage of HBP Fusion Tag from the Target Protein

  Materials (also see protocol 2)
  • HBP‐fusion protein solution (from protocol 2 or protocol 3Alternate Protocol)
  • 1 U/µl bovine thrombin (VWR, cat. no. 95017‐586)
  • 0.2 M PMSF solution
  • Constant temperature water bath
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Figures

Videos

Literature Cited

  Beenken, A., & Mohammadi, M. (2009). The FGF family: Biology, pathophysiology and therapy. Nature Reviews. Drug Discovery, 8, 235–253. doi: 10.1038/nrd2792.
  Capila, I., & Linhardt, R. J. (2002). Heparin‐protein interactions. Angewandte Chemie (International Ed. In English), 41, 391–412. doi: 10.1002/1521‐3773(20020201)41:3%3c390::AID‐ANIE390%3e3.0.CO;2‐B.
  Culley, F. J., Fadlon, E. J., Kirchem, A., Williams, T. J., Jose, P. J., & Pease, J. E. (2003). Proteoglycans are potent modulators of the biological responses of eosinophils to chemokines. European Journal of Immunology, 33, 1302–1310. doi: 10.1002/eji.200323509.
  de Paz, J. L., Moseman, E. A., Noti, C., Polito, L., von Andrian, U. H., & Seeberger, P. H. (2007). Profiling heparin‐chemokine interactions using synthetic tools. ACS Chemical Biology, 2, 735–744. doi: 10.1021/cb700159m.
  Dempewolf, C., Morris, J., Chopra, M., Jayanthi, S., Kumar, T., & Li, W. (2013) Identification of consensus glycosaminoglycan binding strings in proteins, 2013 International Conference on Information Science and Applications (ICISA), pp. 1–5, 24–26.
  Marty, N. J., Rajalingam, D., Kight, A. D., Lewis, N. E., Fologea, D., Kumar, T. K., … Goforth, R. L. (2009). The membrane‐binding motif of the chloroplast signal recognition particle receptor (cpFtsY) regulates GTPase activity. Journal of Biological Chemistry, 284, 14891–14903. doi: 10.1074/jbc.M900775200.
  Mitsi, M., Forsten‐Williams, K., Gopalakrishnan, M., & Nugent, M. A. (2008). A catalytic role of heparin within the extracellular matrix. Journal of Biological Chemistry, 283, 34796–34807. doi: 10.1074/jbc.M806692200.
  Morris, J., Jayanthi, S., Langston, R., Daily, A., Kight, A., McNabb, D. S., … Kumar, T. K. (2016). Heparin‐binding peptide as a novel affinity tag for purification of recombinant proteins. Protein Expression and Purification, 126, 93–103. doi: 10.1016/j.pep.2016.05.013.
  Peysselon, F., & Ricard‐Blum, S. (2014). Heparin‐protein interactions: From affinity and kinetics to biological roles. Application to an interaction network regulating angiogenesis. Matrix Biology, 35, 73–81. doi: 10.1016/j.matbio.2013.11.001.
  Rajalingam, D., Kumar, T. K., & Yu, C. (2005). The C2A domain of synaptotagmin exhibits a high binding affinity for copper: Implications in the formation of the multiprotein FGF release complex. Biochemistry, 44, 14431–14442. doi: 10.1021/bi051387r.
  Raman, R., Sasisekharan, V., & Sasisekharan, R. (2005). Structural insights into biological roles of protein‐glycosaminoglycan interactions. Chemistry & Biology, 12, 267–277. doi: 10.1016/j.chembiol.2004.11.020.
  Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: advances and challenges. Frontiers in Microbiology, 5, 172. doi: 10.3389/fmicb.2014.00172.
  Sivaraja, V., Kumar, T. K., Rajalingam, D., Graziani, I., Prudovsky, I., & Yu, C. (2006). Copper binding affinity of S100A13, a key component of the FGF‐1 nonclassical copper‐dependent release complex. Biophysical Journal, 91, 1832–1843. doi: 10.1529/biophysj.105.079988.
  Tyler‐Cross, R., Sobel, M., Marques, D., & Harris, R. B. (1994). Heparin binding domain peptides of antithrombin III: Analysis by isothermal titration calorimetry and circular dichroism spectroscopy. Protein Science, 3, 620–627. doi: 10.1002/pro.5560030410.
  Wingfield, P. T. (2015). Overview of the Purification of Recombinant Proteins. Current Protocols in Protein Science, 80, 6.1.1–6.1.35. doi: 10.1002/0471140864.ps0601s80.
  Wong, H., & Schotz, M. C. (2002). The lipase gene family. Journal of Lipid Research, 43, 993–999. doi: 10.1194/jlr.R200007‐JLR200.
  Yu, W., & Hill, J. S. (2006). Mapping the heparin‐binding domain of human hepatic lipase. Biochemical and Biophysical Research Communications, 343, 659–665. doi: 10.1016/j.bbrc.2006.02.175.
Key References
  Morris et al. (2016). See above.
  The first detailed report describing the HBP tag.
  Dempewolf et al. (2013). See above.
  Describes the “string” algorithm used to identify heparin binding segments in various proteins.
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