Solubilization and Refolding of Inclusion Body Proteins

Singh, Anupam; Upadhyay, Vaibhav; Panda, Amulya K.

doi:10.1007/978-1-4939-2205-5_15

Cited by 73 publications

(48 citation statements)

References 20 publications

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“…(iii) Purification of inclusion bodies. A literature protocol was adopted (68). The insoluble debris was homogenized in 20 ml of Tris-HCl buffer (50 mM, pH 8.5) containing 1% (wt/wt) deoxycholic acid.…”

Section: Methodsmentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo-and enantioselective ␣,␣-(1¡1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli. To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, K eq , for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDPglucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control. IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.

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Section: Methodsmentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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“…Strains specifically engineered for expression of mammalian proteins were also tried in order to solve this problem. Since none of the methods employed seemed to yield protein in soluble form, we proceeded to continue with BL21 (DE3) and solubilize the GST-sFRP4 using detergents [38,39]. We observed optimum expression of GST-sFRP4 at 28°C after carrying out induction at temperatures ranging from 16 to 37°C.…”

Section: Cloning Expression and Purification Of Sfrp4mentioning

confidence: 95%

“…2c). To obtain the protein in soluble fraction, a number of strategies involving the usage of various detergents were implemented following the literature [38,39]. Finally, 0.32 % of the ionic detergent sarkosyl was used to successfully solubilize the protein from inclusion bodies and mild stirring was done in 1 % Triton X-100.…”

Section: Cloning Expression and Purification Of Sfrp4mentioning

confidence: 99%

Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy

Ghoshal

Ghosh

2016

Mol Cell Biochem

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The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40 %. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated β-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.

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“…The engineering principle developed here should permit the construction of protein-based nanoparticles by the precise sequence manipulation of pre-existing proteins that increase, in a controlled way, the cationic load of the amino terminal regions. Also, the progressive developments in systems and genetic approaches to recombinant protein production 40 increasingly facilitate protein engineering, biofabrication in cell factories 41 and downstream 41,42 . In the current bubbling context of novel protein drugs of interest in cancer therapies and for other conditions 44 , formulating such therapeutic proteins as nanoparticles would offer an interesting engineering tool with a broad applicability in nanomedicine, as the multivalent presentation of homing agents in those materials, either natural or added, is expected to dramatically enhance cell uptake.…”

Section: Discussionmentioning

confidence: 99%

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

Serna

Céspedes

Saccardo

et al. 2016

Nanomedicine: Nanotechnology, Biology and Medicine

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Solubilization and Refolding of Inclusion Body Proteins

Cited by 73 publications

References 20 publications

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy

Rational engineering of single-chain polypeptides into protein-only, BBB-targeted nanoparticles

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