title: Highly purified mussel adhesive protein to secure biosafety for in vivo applications.
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.
authors.forename: Bong-Hyuk
authors.initials: BH
authors.lastname: Choi
authors.forename: Hogyun
authors.initials: H
authors.lastname: Cheong
authors.forename: Yun Kee
authors.initials: YK
authors.lastname: Jo
authors.forename: So Yeong
authors.initials: SY
authors.lastname: Bahn
authors.forename: Jeong Hyun
authors.initials: JH
authors.lastname: Seo
authors.affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea. hjcha@postech.ac.kr.
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004731:Endotoxins / Q000032:analysis / Q000633:toxicity; D004926:Escherichia coli / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004731:Endotoxins; D015850:Interleukin-6; D011506:Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C037403:adhesive protein, mussel
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