RESUMO
Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400-500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.
RESUMO
Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma ractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one hromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.
RESUMO
Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.
Assuntos
Cromatografia de Afinidade/métodos , Cobre/química , Fator VIII/isolamento & purificação , Fator VIII/química , Fator VIII/metabolismo , Humanos , Modelos MolecularesRESUMO
The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.
Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Leptospira interrogans/metabolismo , Glicoproteínas de Membrana/metabolismo , RNA de Transferência de Metionina/metabolismo , Ribonucleases/metabolismo , Sequência de Aminoácidos , Animais , Antitoxinas/química , Antitoxinas/genética , Antitoxinas/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Leptospira interrogans/química , Leptospira interrogans/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Óperon , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.