One-step purification of tissue-type plasminogen activator using affinity chromatography with a special monoclonal antibody under mild conditions.
We have previously isolated a monoclonal antibody, designated as 1-3-1, specific for tissue-type plasminogen activator (t-PA). We have shown that t-PA dissociates from 1-3-1 in the presence of the lysine analogue 6-aminohexanoic acid (6-AHA). Here we describe a method for the one-step immunoaffinity purification of t-PA from conditioned melanoma cell medium, using 1-3-1 immobilised on Sepharose under mild elution conditions, favourable for t-PA. The yield of t-PA (antigen or total protein) from a 1-3-1-Sepharose column, when eluted using a buffer supplemented with 0.2 M 6-AHA at neutral pH, was as effective as other buffers that involve a strong pH-change, i.e., pH 2-3. However, the enzymatic activity of the t-PA purified with 6-AHA was 25 to 30% higher, as compared with t-PA eluted using a pH change. This resulted in a markedly higher specific activity of t-PA purified with 0.2 M 6-AHA, as compared with t-PA purified using a strong pH-change. The purity of t-PA, purified using the present method, was very high, as determined by gel electrophoresis. An additional advantage of the present procedure is that the mild elution conditions prolong the column life.
Bos
R
R
TNO Institute for Ageing and Vascular Research, Gaubius Laboratory, Leiden, Netherlands.
Berger
L
L
Nieuwenhuizen
W
W
D015119:Aminocaproic Acid / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D003470:Culture Media; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008545:Melanoma / Q000378:metabolism; D011136:Polysorbates; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*; D014325:Tromethamine; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003470:Culture Media; D011136:Polysorbates; D014325:Tromethamine; D010959:Tissue Plasminogen Activator; D015119:Aminocaproic Acid