title: Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli.
abstract: BACKGROUND
  Inclusion 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.
  
  
  RESULTS
  Uncontrolled 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Andrea
authors.initials: A
authors.lastname: Castellanos-Mendoza
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Castro-Acosta
authors.forename: Alejandro
authors.initials: A
authors.lastname: Olvera
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Zavala
authors.forename: Miguel
authors.initials: M
authors.lastname: Mendoza-Vera
authors.forename: Enrique
authors.initials: E
authors.lastname: García-Hernández
authors.forename: Alejandro
authors.initials: A
authors.lastname: Alagón
authors.forename: Mauricio A
authors.initials: MA
authors.lastname: Trujillo-Roldán
authors.forename: Norma A
authors.initials: NA
authors.lastname: Valdez-Cruz
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D018533:Biomass; D003224:Congo Red / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000378:metabolism; D013987:Ticks / Q000201:enzymology
chemical_list: D052160:Benzothiazoles; D011994:Recombinant Proteins; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D010727:Phosphoric Diester Hydrolases; C055160:sphingomyelin phosphodiesterase D; D019286:Endopeptidase K; D019791:Guanidine