Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Mallu,Maheswara Reddy, Vemula,Sandeep, Ronda,Srinivasa Reddy
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2016
Materias:	Biological activity Cell lysis Cross flow filtration Fed-batch fermentation Purification Secondary structure
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400012
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:scielo:S0717-34582016000400012
record_format	dspace
spelling	oai:scielo:S0717-345820160004000122016-09-13Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiaeMallu,Maheswara ReddyVemula,SandeepRonda,Srinivasa Reddy Biological activity Cell lysis Cross flow filtration Fed-batch fermentation Purification Secondary structure Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.4 20162016-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400012en10.1016/j.ejbt.2016.06.002
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Biological activity Cell lysis Cross flow filtration Fed-batch fermentation Purification Secondary structure
spellingShingle	Biological activity Cell lysis Cross flow filtration Fed-batch fermentation Purification Secondary structure Mallu,Maheswara Reddy Vemula,Sandeep Ronda,Srinivasa Reddy Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
description	Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.
author	Mallu,Maheswara Reddy Vemula,Sandeep Ronda,Srinivasa Reddy
author_facet	Mallu,Maheswara Reddy Vemula,Sandeep Ronda,Srinivasa Reddy
author_sort	Mallu,Maheswara Reddy
title	Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
title_short	Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
title_full	Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
title_fullStr	Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
title_full_unstemmed	Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae
title_sort	production, purification and characterization of recombinant human antithrombin iii by saccharomyces cerevisiae
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2016
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400012
work_keys_str_mv	AT mallumaheswarareddy productionpurificationandcharacterizationofrecombinanthumanantithrombiniiibysaccharomycescerevisiae AT vemulasandeep productionpurificationandcharacterizationofrecombinanthumanantithrombiniiibysaccharomycescerevisiae AT rondasrinivasareddy productionpurificationandcharacterizationofrecombinanthumanantithrombiniiibysaccharomycescerevisiae
_version_	1718441933058080768

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Ejemplares similares