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.

New tools for recombinant protein production in Escherichia coli: A 5‐year update

“…The NT‐11 tag enhanced the soluble production of all tested proteins, which were also His‐tagged. Moving up to fusion tags of higher molecular mass, the Kumar lab designed a 34‐amino acid heparin‐binding affinity tag (HB‐tag) . HB‐tagged proteins can be purified using a heparin‐agarose resin under naturing or denaturing conditions.…”

Guidelines to reach high-quality purified recombinant proteins

“…Costa et al 2014;Yadav et al 2016). Nevertheless, novel tags, from natural origin (Li et al 2016) or rationally designed (Morris et al 2016), are continually being reported in the literature. Among the affinity tags recently described, interesting alternatives to the His-tag have been presented (Vargas-Cortez et al 2016;Cantu-Bustos et al 2016).…”

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding

“…Some of the common putative heparin-binding segments are XBBXBX, XBBBXXBX, and XBBXXBBBXXBBX, where B is one of the three basic amino acids (mainly arginine and lysine) and X is any of the other 17 natural amino acids. These heparin binding segments function by electrostatic interaction with the negatively charged sulfate and carboxyl groups in heparin or heparan sulfate [19].…”