Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA).
Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.
Vlakh
E
E
Institute of Macromolecular Compounds, Russian Academy of Sciences, Bolshoy pr. 31, 199004 St. Petersburg, Russia.
Ostryanina
N
N
Jungbauer
A
A
Tennikova
T
T
D000818:Animals; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D006224:Cricetinae; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010960:Plasminogen Activators / Q000235:genetics / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*
D010455:Peptides; D011108:Polymers; D011994:Recombinant Proteins; D010960:Plasminogen Activators