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Large scale, rapid purification of recombinant tissue‐type plasminogen activator
Author(s) -
Dodd I.,
Jalalpour S.,
Southwick W.,
Newsome P.,
Browne M.J.,
Robinson J.H.
Publication year - 1986
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(86)81075-4
Subject(s) - chromatography , chemistry , plasminogen activator , sepharose , coomassie brilliant blue , recombinant dna , polyacrylamide gel electrophoresis , staining , biochemistry , biology , enzyme , gene , genetics , endocrinology
Recombinant tissue‐type plasminogen activator (rt‐PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate‐Sepharose CL4B and lysine‐Sepharose CL4B. After eight successive purifications the rt‐PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 × 10 6. Analysis by SDS‐polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at M r = 63000 and 65000; most of the material was in the 1‐chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.