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).
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.
Kang
Kwan-Yub
KY
Laboratory of Pharmaceutical Technology, Mogam Biotechnology Research Institute, 341, Pojung-ri, Guseong-eup, Yongin-city, Kyonggi-do 449-913, South Korea.
Kim
Sung-Geun
SG
Kim
Won-Kyung
WK
You
Hyun-Kyung
HK
Kim
Young-Jo
YJ
Lee
Jong-Hyuk
JH
Jung
Kyung-Hwan
KH
Kim
Chan-Wha
CW
D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001054:Apolipoproteins A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005340:Fibrinogen / Q000737:chemistry / Q000302:isolation & purification; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D006801:Humans; D066298:In Vitro Techniques; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018082:Kringles / Q000235:genetics*; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013997:Time Factors
D020533:Angiogenesis Inhibitors; D001054:Apolipoproteins A; D011994:Recombinant Proteins; D005340:Fibrinogen; D008239:Lysine