An approach for the stable immobilization of proteins

Deborah Leckband, Robert Langer

Research output: Contribution to journalArticle

Abstract

An approach is presented for the stable covalent immobilization of proteins with a high retention of biological activity. First, chemical modification studies were used to establish enzyme structural and functional properties relevant to the covalent immobilization of an enzyme to agarose based supports. Heparinase was used as a model enzyme in this set of studies. Amine modifications result in 75-100% activity loss, but the effect is moderated by a reduction in the degree of derivatization. N-hydroxysuccinimide, 1,1,1-trifluoroethanesulfonic acid, and epoxide activated agarose were utilized to determine the effect of amine reactive supports on immobilized enzyme activity retention. Cysteine modifications resulted in 25-50% loss in activity, but free cysteines were inaccessible to either immobilized bromoacetyl or p-chloromercuribenzoyl groups. Amine reactive coupling chemistries were therefore utilized for the covalent immobilization of heparinase. Second, to ensure maximal stability of the immobile protein-support linkage, the identification and subsequent elimination of the principal sources of protein detachment were systematically investigated. By using high-performance liquid chromatography (HPLC), electrophoresis and radiolabeling techniques, the relative contributions of four potential detachment mechanisms - support degradation, proteolytic degradation, desorption of noncovalently bound protein, and bond solvolysis - were quantified. The mechanisms of lysozyme, bovine serum albumin, and heparinase leakage from N-hydroxysuccinimide or 1,1,1-trifluoroethanesulfonic acid activated agarose were elucidated. By use of stringent postimmobilization support wash procedures, noncovalently bound protein was shown to be the predominant source of immobilized protein loss. An effective postimmobilization washing procedure is presented for the removal of adsorbed protein and the complete elimination of immobilized protein loss.

Original languageEnglish (US)
Pages (from-to)227-237
Number of pages11
JournalBiotechnology and bioengineering
Volume37
Issue number3
DOIs
StatePublished - Feb 5 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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