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

Castellanos-Mendoza, Andrea; Castro-Acosta, Ricardo M.; Olvera, Alejandro; Zavala, Guadalupe; Mendoza-Vera, Miguel; García‐Hernández, Enrique; Alagón, Alejandro; Trujillo‐Roldán, Mauricio A.; Valdez‐Cruz, Norma A.

doi:10.1186/preaccept-1104793932132453

Cited by 7 publications

(17 citation statements)

References 85 publications

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“…In this contribution we introduce nano particle tracking analysis (NTA) as method to analyze and size a large number of biologic nano particles individually. For biologic particles the dynamic range of NTA spans 100–1500 nm, which fits the reported size range of IBs from 170 to 1300 nm . NTA uses a laser as light source, which passes through the sample particle suspension and illuminates the particles.…”

Section: Introductionsupporting

confidence: 53%

“…Besides sample preparation, the method of sample fixation has been a topic of vivid discussion. A common approach for sample fixation of IBs is chemical fixation or air drying of the specimen on a copper grid . Despite the wide usage of these methods, the specimen might be altered, subcellular structures can condense and shrinkage can occur.…”

Section: Discussionmentioning

confidence: 99%

“…The increased volume in turn calls for bigger column diameters of economically expensive DSP purification columns . Recent developments have enabled the concise measurement of solubility in respect to time as well as in respect to the concentration of chaotrope reagents .…”

Section: Introductionmentioning

confidence: 99%

“…Dynamic light scattering (DLS) has extensively been utilized to size biological nano particles . Nevertheless, since this method only measures one variable its sensitivity is greatly impaired by multimodal distributions as well as by background particles .…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, since this method only measures one variable its sensitivity is greatly impaired by multimodal distributions as well as by background particles . As a counteraction, sample purification by serial washing steps or full‐grown purification techniques as ultracentrifugation have been investigated but these measures increase the risk of a measurement bias caused by sample preparation.…”

Section: Introductionmentioning

confidence: 99%

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High throughput inclusion body sizing: Nano particle tracking analysis

Reichelt

Kaineder

Brillmann

et al. 2017

Biotechnology Journal

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The expression of pharmaceutical relevant proteins in Escherichia coli frequently triggers inclusion body (IB) formation caused by protein aggregation. In the scientific literature, substantial effort has been devoted to the quantification of IB size. However, particle-based methods used up to this point to analyze the physical properties of representative numbers of IBs lack sensitivity and/or orthogonal verification. Using high pressure freezing and automated freeze substitution for transmission electron microscopy (TEM) the cytosolic inclusion body structure was preserved within the cells. TEM imaging in combination with manual grey scale image segmentation allowed the quantification of relative areas covered by the inclusion body within the cytosol. As a high throughput method nano particle tracking analysis (NTA) enables one to derive the diameter of inclusion bodies in cell homogenate based on a measurement of the Brownian motion. The NTA analysis of fixated (glutaraldehyde) and non-fixated IBs suggests that high pressure homogenization annihilates the native physiological shape of IBs. Nevertheless, the ratio of particle counts of non-fixated and fixated samples could potentially serve as factor for particle stickiness. In this contribution, we establish image segmentation of TEM pictures as an orthogonal method to size biologic particles in the cytosol of cells. More importantly, NTA has been established as a particle-based, fast and high throughput method (1000-3000 particles), thus constituting a much more accurate and representative analysis than currently available methods.

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Section: Introductionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

High throughput inclusion body sizing: Nano particle tracking analysis

Reichelt

Kaineder

Brillmann

et al. 2017

Biotechnology Journal

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Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Zhang

2019

J of Chemical Tech & Biotech

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BACKGROUND: Recombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a-pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high-level soluble pod production. RESULTS:During recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L −1 at pH 6.0 after 64 h induction. CONCLUSION: Soluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance.

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Perspectives of inclusion bodies for bio-based products: curse or blessing?

Slouka

Kopp

Spadiut

et al. 2018

Appl Microbiol Biotechnol

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The bacterium Escherichia coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body-based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.

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

Cited by 7 publications

References 85 publications

High throughput inclusion body sizing: Nano particle tracking analysis

High throughput inclusion body sizing: Nano particle tracking analysis

Soluble recombinant pyruvate oxidase production in Escherichia coli can be enhanced and inclusion bodies minimized by avoiding pH stress

Perspectives of inclusion bodies for bio-based products: curse or blessing?

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