Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli.
The most frequently occurring kringle 4 domain of human apolipoprotein (a), Kringle 4-subtype 2 (K4(2)), was expressed as a fusion protein with the maltose binding protein in Escherichia coli using the "tac" promoter. Although the fusion protein was expressed without a signal sequence, 25% was secreted into the periplasmic space; the remainder was found associated with the soluble cytosolic fraction. The fusion protein was readily isolated from whole cell lysate by amylose agarose affinity chromatography. Although a factor Xa cleavage site was engineered into the fusion protein, it was found that release of the K4(2) protein was most conveniently achieved by proteolysis with subtilisin A. The cleavage product produced in this way was shown to be intact K4(2) with only the first three amino acid residues of the leading flanking peptide missing, as judged by N-terminal sequence analysis. K4(2) was isolated from the hydrolysate by FPLC on a Mono-Q column with a yield of 170 +/- 30 micrograms/g wet cells. The resulting protein was monomeric in phosphate-buffered saline as judged by size-exclusion chromatography and appeared to be folded as shown by spectroscopic and immunological assays. The recombinant K4(2) did not bind to either lysine- or proline-Sepharose, suggesting that the ligand binding activities of lipoprotein (a) may reside in the other kringle domains of apolipoprotein (a).
Li
Z
Z
Department of Medicine, University of Chicago, Illinois 60637.
Gambino
R
R
Fless
G M
GM
Copeland
R A
RA
Halfpenny
A J
AJ
Scanu
A M
AM
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001053:Apolipoproteins / Q000235:genetics / Q000302:isolation & purification*; D053241:Apoprotein(a); D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D017270:Lipoprotein(a); D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification*; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives
D018528:ATP-Binding Cassette Transporters; D001053:Apolipoproteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D017270:Lipoprotein(a); C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C030675:lysine-sepharose; C461599:maltose transport system, E coli; D012685:Sepharose; D053241:Apoprotein(a); D015951:Factor Xa