Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing

Anders Heebøll-Nielsen, W.S. Choe, A.P.J. Middelberg, Owen R. T. Thomas

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving W chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K-d similar to 0.08 muM) was exhibited by Cu2+-charged type II support materials. Equilibrium adsorption of Ll to these nonporous supports was achieved very rapidly ( 100 mM imidazole in the equilibration buffer. The influence of feedstock complexity on Ll adsorption to the Cu2+-charged type II magnetic chelators was studied using various dilutions of four crude chemical E. coli cell extracts containing denatured L I protein. Undiminished Ll adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L-1) and spermine-treated chemical cell extract (feedstock B). Efficient recovery of Ll from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu2+-chelator particles following batch adsorption of L1. Over 70% of the initial Ll present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of similar to10.
    Original languageEnglish
    JournalBiotechnology Progress
    Volume19
    Issue number3
    Pages (from-to)887-898
    ISSN8756-7938
    Publication statusPublished - 2003

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