Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor

Dae Young Kim, Jeewon Lee, Vibhor Saraswat, Young Hoon Park

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The specific molecular interactions of α-helical peptide, human glucagon (i.e., intermolecular self-association and specific receptor-binding affinity) provided a rationale for using the glucagon as the fusion expression partner to achieve high productivity of foreign proteins both in vivo (in bacterial fusion-expression system) and in vitro (in affinity column chromatography). The fusion of glucagon peptide(s) effectively promoted homogeneous aggregate formation of recombinant proteins while avoiding intermolecular crosslinking by disulfide bridges. High sensitivity of the self-aggregation to sequence effects resulted from two distinct nonpolar domains of glucagon, determining specificity of molecular interaction and aggregate size of recombinant proteins. An N-terminal domain of glucagon molecule (Phe6-Tyr10-Tyr13) could be a certain hydrophobic moiety involved in intermolecular self-association (probably, via helix-helix docking), while a C-terminal domain (Phe22-Trp25-Leu26) seems to critically affect the oligomer size in the off-pathway aggregation of synthesized fusion proteins. An N-terminal extracellular domain of human glucagon receptor was recombinantly expressed in Escherichia coli, immobilized to a chromatography column, and efficiently renatured to a conformation that attains high specificity in interaction with N-terminus glucagon molecules of recombinant fusion proteins. Through column chromatography employing the receptor fragment as affinity ligand, the recombinant proteins were efficiently purified from total intracellular proteins, and the long-term ligand stability was evidently proven through multiple cyclic-purification experiments. Major scaffolds for using protein ligands are large-scale production in a low-cost expression system and long-term stable operation with selective-binding affinity. From this point of view, the extracellular fragment of human glucagon receptor used in this study seems to be a new potent ligand for fusion protein-based affinity chromatography.

Original languageEnglish
Pages (from-to)418-428
Number of pages11
JournalBiotechnology and Bioengineering
Volume69
Issue number4
Publication statusPublished - 2000 Aug 20
Externally publishedYes

Fingerprint

Glucagon Receptors
Recombinant proteins
Glucagon
Recombinant Proteins
Escherichia coli
Purification
Fusion reactions
Association reactions
Proteins
Column chromatography
Ligands
Affinity chromatography
Molecular interactions
Affinity Chromatography
Peptides
Chromatography
Agglomeration
Recombinant Fusion Proteins
Molecules
Scaffolds (biology)

Keywords

  • Affinity purification
  • Fusion protein
  • Glucagon
  • Receptor
  • Self-association

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor. / Kim, Dae Young; Lee, Jeewon; Saraswat, Vibhor; Park, Young Hoon.

In: Biotechnology and Bioengineering, Vol. 69, No. 4, 20.08.2000, p. 418-428.

Research output: Contribution to journalArticle

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