Highly purified mussel adhesive protein to secure biosafety for in vivo applications

Title
Highly purified mussel adhesive protein to secure biosafety for in vivo applications
Author(s)
서정현최봉혁[최봉혁]정호균[정호균]조윤기[조윤기]반소영[반소영]차형준[차형준]
Keywords
ENDOTOXIN REMOVAL; ESCHERICHIA-COLI; MYTILUS-EDULIS; TRITON X-114; RECOMBINANT; EXPRESSION
Issue Date
201404
Publisher
BIOMED CENTRAL LTD
Citation
MICROBIAL CELL FACTORIES, v.13
Abstract
Background: Unique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult. Results: In the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151. Conclusions: Through this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.
URI
http://hdl.handle.net/YU.REPOSITORY/32562http://dx.doi.org/10.1186/1475-2859-13-52
ISSN
1475-2859
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공과대학 > 화학공학부 > Articles
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