Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V)

Kwan Yub Kang, Sung Geun Kim, Won Kyung Kim, Hyun Kyung You, Young Jo Kim, Jong Hyuk Lee, Kyung Hwan Jung, Chan Wha Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Original languageEnglish
Pages (from-to)216-225
Number of pages10
JournalProtein Expression and Purification
Volume45
Issue number1
DOIs
Publication statusPublished - 2006 Jan 1

Fingerprint

Kringles
Apoprotein(a)
Chromatography
Escherichia coli
Purification
Inclusion Bodies
High Pressure Liquid Chromatography
Fibrinogen
Lysine
Native Polyacrylamide Gel Electrophoresis
Urea
Ion exchange
Ion Exchange Chromatography
Reverse-Phase Chromatography
Oxidation
acetonitrile
Oxidation-Reduction

Keywords

  • Angiogenesis
  • Apolipoprotein (a)
  • Kringle
  • Refolding

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli : Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V). / Kang, Kwan Yub; Kim, Sung Geun; Kim, Won Kyung; You, Hyun Kyung; Kim, Young Jo; Lee, Jong Hyuk; Jung, Kyung Hwan; Kim, Chan Wha.

In: Protein Expression and Purification, Vol. 45, No. 1, 01.01.2006, p. 216-225.

Research output: Contribution to journalArticle

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