Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector

Li, Jianghui; Han, Qinxia; Zhang, Tao; Du, Jing; Sun, Qianqian; Pang, Yilin

doi:10.1016/j.btre.2018.e00261

Cited by 9 publications

(8 citation statements)

References 30 publications

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“…Proteins with low structural stability experience a high risk of degradation, causing insufficient proteins to be produced ( 46 ). Many proteins are expressed with tags or fusion partners to prevent proteolytic degradation and increase protein stability by promoting correct protein folding ( 47 – 49 ).…”

Section: Resultsmentioning

confidence: 99%

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides

Ikeno

Almansoori

et al. 2022

Microbiol Spectr

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Section: Resultsmentioning

confidence: 99%

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides

Ikeno

Almansoori

et al. 2022

Microbiol Spectr

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“…The recombinant plasmids were constructed as previously described ( Yang et al, 2015 ; Li et al, 2018 ). The iscS gene was amplified from the E. coli MC4100 genome.…”

Section: Methodsmentioning

confidence: 99%

“…CD activity assay was conducted as previously described ( Siegel, 1965 ; Yang et al, 2015 ; Li et al, 2018 ). Briefly, purified recombinant CDs (5 μM) were incubated with buffer A in the presence of 3 mM dithiothreitol (DTT) for 5 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…NFS1 and ISD11 are mitochondrial proteins ( Friemel et al, 2017 ), and mutations in these proteins can lead to rare but severe mitochondrial disorders ( Farhan et al, 2014 ; Saha et al, 2015 ). Additionally, functionally producing these proteins in E. coli is particularly challenging because majority of the over-expressed protein is sequestered in inclusion bodies ( Marelja et al, 2008 ; Li et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

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Roles of conserved active site residues in the IscS cysteine desulfurase reaction

Pang

Wang²,

Gao³

et al. 2023

Front. Microbiol.

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Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5′-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography–tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

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“…SUMO is a ubiquitin-like protein (Johnson and Hochstrasser 1997) of approximately 11 kDa that is frequently used to improve target protein solubility and stability through N-terminal fusion (Bird 2011). The SUMO tag can be cleaved from the chimeric protein by Ulp1 protease, which specifically recognizes the SUMO tertiary structure, to generate the native protein without any redundant amino acids (Li et al 2018). In light of the high efficiency of the SUMO-Ulp1 system in protein purification, we constructed two different vectors for the expression of SUMO-fused recombinant protein and Ulp1 protease on the surfaces of E. coli cells.…”

Section: Introductionmentioning

confidence: 99%

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease

et al. 2020

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The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ionexchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.

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Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector

Cited by 9 publications

References 30 publications

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides

Characterization of Sphingobacterium sp. Ab3 Lipase and Its Coexpression with LEA Peptides

Roles of conserved active site residues in the IscS cysteine desulfurase reaction

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease

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