Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more c...

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Autores principales:	Ping,Ruo, Sin Teoh,Leong, Kwai Chan,Mooi, Miswan,Noorizan, Yin Khoo,Boon
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2016
Materias:	Cell density Pichia pastoris Total protein concentration Recombinant protein expression
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000300001
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spelling	oai:scielo:S0717-345820160003000012016-07-06Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastorisPing,RuoSin Teoh,LeongKwai Chan,MooiMiswan,NoorizanYin Khoo,Boon Cell density Pichia pastoris Total protein concentration Recombinant protein expression Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.19 n.3 20162016-05-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000300001en10.1016/j.ejbt.2016.01.007
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Cell density Pichia pastoris Total protein concentration Recombinant protein expression
spellingShingle	Cell density Pichia pastoris Total protein concentration Recombinant protein expression Ping,Ruo Sin Teoh,Leong Kwai Chan,Mooi Miswan,Noorizan Yin Khoo,Boon Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
description	Background: Human is an essential cellular enzyme that is found in all human cells. As this enzyme is upregulated in cancer cells exceedingly, it is used as a target for cancer chemotherapeutic drug development. As such, producing the in-house enzyme for the purpose to speed up the search for more cost-effective and target specific hTopoI inhibitors is warranted. This study aims to compare the optimised conditions for the expression of hTopoI in KM71H (MutS) and X33 (Mut+) strains of Pichia pastoris. P. pastoris transfected with an hTopoI recombinant vector was used for the optimization of a higher level of hTopoI expression. Results: In the process, fed-batch cultivation parameters that influence the expression of hTopoI, such as culture temperature, methanol induction and feeding strategy, were optimised in the transfected KM71H and X33 P. pastoris strains in a shake flask system. The cell density and total protein concentration (protein level) of transfected P. pastoris were compared to determine the optimum culture conditions for each transfected P. pastoris strain. A higher hTopoI level was observed in the transfected KM71H culture supernatant (2.26 ng/mL) when the culture was incubated in the optimum conditions. Conclusions: This study demonstrated that MutS strain (KM71H) expressed and secreted a higher level of hTopoI heterologous protein in the presence of methanol compared to the Mut+ strain; X33 (0.75 ng/mL). However, other aspects of optimization, such as pH, should also be considered in the future, to obtain the optimum expression level of hTopoI in P. pastoris.
author	Ping,Ruo Sin Teoh,Leong Kwai Chan,Mooi Miswan,Noorizan Yin Khoo,Boon
author_facet	Ping,Ruo Sin Teoh,Leong Kwai Chan,Mooi Miswan,Noorizan Yin Khoo,Boon
author_sort	Ping,Ruo
title	Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
title_short	Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
title_full	Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
title_fullStr	Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
title_full_unstemmed	Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris
title_sort	comparing the expression of human dna topoisomerase i in km71h and x33 strains of pichia pastoris
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2016
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000300001
work_keys_str_mv	AT pingruo comparingtheexpressionofhumandnatopoisomeraseiinkm71handx33strainsofpichiapastoris AT sinteohleong comparingtheexpressionofhumandnatopoisomeraseiinkm71handx33strainsofpichiapastoris AT kwaichanmooi comparingtheexpressionofhumandnatopoisomeraseiinkm71handx33strainsofpichiapastoris AT miswannoorizan comparingtheexpressionofhumandnatopoisomeraseiinkm71handx33strainsofpichiapastoris AT yinkhooboon comparingtheexpressionofhumandnatopoisomeraseiinkm71handx33strainsofpichiapastoris
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Comparing the expression of human DNA topoisomerase I in KM71H and X33 strains of Pichia pastoris

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