A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein

Chen, Zhenya; Zhao, Luyao; Ru, Jiakang; Yu, Shengzhu; Yu, Huan; Ren, Huiyong; Zhang, Yuhong; Zhang, Wei; Lin, Fuchang; Huo, Yi-Xin

doi:10.1016/j.jbiotec.2019.06.002

Cited by 4 publications

(9 citation statements)

References 44 publications

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“…Importantly, the linker GSGSGS inserted into the fusion protein does not disrupt the catalytic properties of any protein in the fusion protein. 29 Therefore, we assumed that the linear sequences of Kivd and LeuDH can fold properly when they are incorporated in the fusion protein. The assessments by tFold, TrRosetta, and Modeler 10.1 also confirmed their ability to fold correctly during the rational design of this fusion protein.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Genes cipA (UNIPROT code: Q7N6H4) and eGFP (NCBI Accession: AHK23750.1) were amplified from plasmid pET-28a-cipA-dnaB-eGFP. 29 The primers sequences for amplifying kivd, leuDH, cipA, and eGFP were kivd-F (5′atgtatacagtaggagattacctattagaccgatt-3′), kivd-R (5′-tgatttattttgttcagcaaatagtttgccca-3′), leuDH-F (5′-atgaaaatcttcgattacatggaaaaatatgat-3′), leuDH-R (5′-tttgttgttaaaattgatcaggttgcgttgatc-3′), cipA-F (5′-atgatcaacgacatgcacccgtctctgatcaa-3′), cipA-R (5′-atagagatttcaacgcagttgatgtagtcgc-3′), eGFP-F (5′-atggtgagcaagggcgaggagctgttcaccgg-3′), and eGFP-R (5′-cttgtacagctcgtccatgccgagagtgatccc-3′). The rf p gene (NCBI accession: ACD13196.1) was synthesized by overlap extension polymerase chain reaction (OE-PCR).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…The voltage was first set at 80 V for 30 min and then 120 V for 1 h. Subsequently, the gel was transferred into the staining solution to stain for 45 min. Then, the gel was transferred into the destaining solution to destain overnight 29. PCIs Conversion Capacity and Thermal Stability Measurements.…”

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confidence: 99%

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In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy

Zhao

Chen

Lin

et al. 2021

J. Agric. Food Chem.

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The in vitro biosynthesis of high-value compounds has become popular and attractive. The convenient and simple strategy of enzyme immobilization has been significant for continuous and efficient in vitro biosynthesis. On the basis of that, this work established a one-step self-assembly-based immobilization strategy to efficiently biosynthesize isobutyraldehyde in vitro. Isobutyraldehyde is a crucial precursor for the synthesis of foods and spices. The established CipA scaffold-based strategy can express and immobilize enzymes at the same time, and purification requires only one centrifugation step. Structural simulations indicated that this scaffold-dependent self-assembly did not influence the structure or catalytic mechanisms of the isobutyraldehyde production-related enzymes leucine dehydrogenase (LeuDH) and ketoisovalerate decarboxylase (Kivd). Immobilized LeuDH and Kivd displayed a higher conversion capacity and thermal stability than the free enzymes. Batch conversion experiments demonstrated that the recovered immobilized LeuDH and Kivd have similar conversion capacities to the enzymes used in the first round of reaction. The continuous production of isobutyraldehyde was achieved by filling the immobilized enzymes into the column of a constructed device. This study not only expands the application range of self-assembly systems but also provides guidance for the in vitro production of value-added compounds.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

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In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy

Zhao

Chen

Lin

et al. 2021

J. Agric. Food Chem.

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“…N pro is a highly hydrophobic protein with 168 residues [2], which has been used as an aggregating tag to increase the expression level of target protein in the form of inclusion body. Some short self-assembling peptide tags such as ELK16, L 6 KD and GFIL8 [3] and protein tags like CipA [4] can also induce protein aggregates. Unlike conventional inclusion bodies, these proteins are highly active.…”

Section: Introductionmentioning

confidence: 99%

“…Non-chromatographic purification based on aggregating tags reduces the purification processes and the experimental costs at laboratory scale. However, the self-cleaving inteins such as △I-CM, DnaE and DnaB should be co-expressed with aggregating tags for selfcleaving, induced by pH shift and/or temperature changes to remove the aggregating tags and restore the target proteins to the soluble phase [4,5].…”

Section: Introductionmentioning

confidence: 99%

mem-iLID, a fast and economic protein purification method

et al. 2021

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Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

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Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli

Amaranto

Vaccarello

Correa

et al. 2021

Journal of Biotechnology

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A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein

Cited by 4 publications

References 44 publications

In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy

In Vitro Biosynthesis of Isobutyraldehyde Through the Establishment of a One-Step Self-Assembly-Based Immobilization Strategy

mem-iLID, a fast and economic protein purification method

Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli

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