Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

Microbial Cell Factories

Research

Mauricio A Trujillo-Roldán¹  Norma A Valdez-Cruz¹  Andrea Castellanos-Mendoza¹  Ricardo M Castro-Acosta²  Alejandro Olvera²  Alejandro Alagón²  Miguel Mendoza-Vera²  Enrique García-Hernández³  Guadalupe Zavala⁴ 

[1] Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP. 70228, México, CP. 04510, Mexico D.F, México;Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor., Mexico;Instituto de Química, Universidad Nacional Autónoma de México, Mexico D.F, Mexico;Unidad de Microscopía. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor., Mexico;

关键词: Inclusion bodies;    Culture conditions;    pH;    E. coli;    β-structure;    Sphingomyelinase-D;    Recombinant proteins;   

DOI  :  10.1186/s12934-014-0137-9

 received in 2014-06-12, accepted in 2014-09-04,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundInclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies.ResultsUncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding.ConclusionsThe manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

【 授权许可】

Unknown   
© Castellanos-Mendoza et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.