title: Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.
abstract: Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Morris
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Jayanthi
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Rebekah
authors.initials: R
authors.lastname: Langston
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Daily
authors.affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Alicia
authors.initials: A
authors.lastname: Kight
authors.affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: David S
authors.initials: DS
authors.lastname: McNabb
authors.affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, USA.
authors.forename: Ralph
authors.initials: R
authors.lastname: Henry
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA. Electronic address: sthalla@uark.edu.
authors.forename: Thallapuranam Krishnaswamy Suresh
authors.initials: TKS
authors.lastname: Kumar
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006493:Heparin / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: C106404:ALBINO 3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D006493:Heparin
keywords: Affinity tag; Heparin; Recombinant proteins and protein purification; Single-step