Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.
A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.
DeSerrano
V S
VS
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Menhart
N
N
Castellino
F J
FJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator; D008239:Lysine