Abstract
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. 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. 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.
Highlights
Bacteria like E. coli have been a successful cellular model to produce useful recombinant proteins in modern biotechnology [1]
By controlling the external pH at 7.5 ± 0.1, the cytoplasmic pH is maintained in the same range simulating the physiological E. coli conditions [56]
No lag phase was found in both cultures, but significant differences were observed in the specific growth rate: 1.34 ± 0.06 h−1 in uncontrolled pH cultures, and 1.21 ± 0.04 h−1 in controlled pH conditions
Summary
Bacteria like E. coli have been a successful cellular model to produce useful recombinant proteins in modern biotechnology [1]. When heterologous protein over-expression occurs, an inefficient folding could occur, which together with the shortage of chaperones may promote protein aggregation [2,3] Those aggregates are called inclusion bodies [4,5], and can be formed in the cytoplasmic or periplasmic area [6,7]. The IB formation and its maintenance involve a complex network of intracellular responses related to culture conditions, leading to complex and stable structures sometimes showing bioactivity [13,22] Due to their different physicochemical properties, IBs have been proposed for various uses, such as catalysts, support materials, drug delivery agents, cell therapy, and immunogens, and their recent application has become an important new topic in biology, medicine and biotechnology [11,23,24,25,26,27,28,29,30]. 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
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