Plasminogen separation

Vlakh E, Ostryanina N, Jungbauer A, and Tennikova T. Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA). J. Biotechnol. 2004 107 275-284. 

Applications of CIEF for the separation of isoforms of transferrin have been reported by several groups [1,2,5]. Transferrin contains different number of sialic add residues, with an additional -1 charge added per residue. Also, transferrin bound to different amount of iron atoms has been separated by CIEF [1,2,5]. Glyco-forms of recombinant tissue-type plasminogen activa-... 

Figure 10.13 shows the excellent performance of reversed-phase HPLC in biotechnological research. It represents the separation of the tryptic hydrolysate of the normal form and of a mutant of tissue-type plasminogen activator. This protein is built up of 527 amino acids and has a mass of approximately 67 000 Da. The mutant differs in a single amino acid, which leads to a deviating retention time of this specific fragment. 

Figure 10.13 Separation of the tryptic hydrolysate of tissue-type plasminogen activator [reproduced with permission from R.L. Garnick, N.J. Solli and P.A. Papa, Anal. Chem., 60,2546 (1988)]. Conditions stationary phase. Nova PakClS, 5 pm mobile phase Imlrnin" 50 mM sodium phosphate pH 2.8-acetonitrile, step gradient UV detector 210 nm. Top the normal protein with arginine at position 275 below the mutant with glutamic acid at position 275.

Wu S L (1997). The use of sequential high-performance liquid chromatography and capillary zone electrophoresis to separate the glycosylated peptides from recombinant tissue plasminogen activator to a detailed level of microheterogeneity. Analyt. Biochem. 253 85-97. 

The fibrin indicator gel was prepared as outlined in the schematic (Fig. 1) and as described previously (R4, R5). Briefly, plasminogen-rich fibrinogen was slowly solubilized at a concentration of 5mg/ml in 0.85% (w/v) saline (prewarmed to 37 °C) with gentle mixing (inversion several times over 1.5-2 h) in a temperature-controlled water bath at 37 °C. Slow solubilization of fibrinogen was necessary to prevent protein flocculation. Agarose was separately prepared as a 1% (w/v) solution in phosphate-buffered saline (PBS, pH... 

Moorhouse, K.G., Rickel, C.A., and Chen, A.B., Electrophoretic separation of recombinant tissue-type plasminogen activator glycoforms Validation issues for capillary isoelectric focusing methods, Electrophoresis, 17, 423, 1996. 

Several pure proteins have been purified and checked for their purity, microheterogeneity, or diagnostic significance by CE. In this case, coated capillaries are preferred for this separation. Many of the proteins can be analyzed by CE however, sensitive detectors such as fluorescence are necessary [68-70]. Examples of proteins studied by CE separately or in a profile are transferrin isoforms, which are important as markers of alcoholism [71], a-1 antitrypsin [72], recombinant human erythropoietin glycoforms that stimulates erythopiosis [73], plasminogen tissue activator [74], prions [75], urothelial carcinoma proteins in urine [76,77], and numerous urinary proteins [78]. 

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