Cleavage of fusion proteins on the affinity resins using the TEV protease variant

Zhu, Kejun; Zhou, Xuan; Yan, Yanping; Mo, Hongmei; Xie, Yifan; Cheng, Beijiu; Fan, Jun

doi:10.1016/j.pep.2016.02.003

Cited by 16 publications

(7 citation statements)

References 32 publications

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“…5F, lane 3), but another band representing the His6-hanA1 was indicated, based on the molecular mass. The co-elution of the fusion tags and other proteins via on-resin cleavage is also identi ed by our previous reports [24,51].…”

Section: A Nity Puri Cation and On-resin Cleavage Of The Hana1-emgfp ...mentioning

confidence: 78%

Investigating the applicability of human annexin A1 as an affinity tag for separation and purification of the target proteins

Zhang

Dang

et al. 2022

Preprint

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Backgrounds Rapid separation methods with fusion of the affinity tags have been developed. However, the affinity tag for simply and cheaply separating the fused target protein is still lacking. Results Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli cytoplasm were optimized via precipitation with CaCl2 and re-solubilization with EDTA-Na2. Combination of the affinity precipitation with each of three affinity purification approaches increased the hanA1-EmGFP purity, and on-resin cleavage facilitated release of tag-free EmGFP. With addition of Triton X-100 to the culture, the fused EmGFP, red fluorescent protein mCherry, or the FMN-dependent fluorescent protein LOV, was also prepared with increasing CaCl2concentrations after it was secreted to the culture from E. coli, whereas the hanA1-EmGFP fused to the amyQ signal sequence was insolubly produced in Bacillus subtilis cells, and the hanA1-EmGFP fused to the α signal peptide produced in Pichia pastoris was unable to be secreted to the culture. The affinity separation was visualized by use of three fluorescent proteins including the EmGFP, mCherry and LOV, and two colored proteins including a bacterial hemoglobin, and maize sirohydrochlorin ferrochelatase (mSF) showing brown containing the [2Fe–2S] cluster. The added EDTA-Na2disrupted the mSF structure, and inhibited activities of the selected four metal-dependent enzymes, but showed little impact on two specific proteases for cleaving the fusion proteins. After affinity precipitation, the tagged lysine decarboxylase was prepared as cross-linked enzyme aggregates. Conclusion The hanA1 tag is ideal for simple, rapid and cost-effective separation of the target proteins via intracellular and extracellular production in E. coli. This tag is also used for further affinity purification of the selected proteins and enzymes potentially applied in industry and diagnosis.

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Section: A Nity Puri Cation and On-resin Cleavage Of The Hana1-emgfp ...mentioning

confidence: 78%

Investigating the applicability of human annexin A1 as an affinity tag for separation and purification of the target proteins

Zhang

Dang

et al. 2022

Preprint

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“…Reduce the amount of time the protein is incubating with the beads. Alternatively, use a different elution method, such as removal of the affinity tag by site-specific proteolysis (Zhu et al, 2017).…”

Section: Understanding Resultsmentioning

confidence: 99%

Isolation of Protein Complexes from Tobacco Leaves by a Two‐Step Tandem Affinity Purification

2022

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Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non‐model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein‐packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two‐step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single‐particle cryo‐electron microscopy—we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1‐XopQ complex

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“…Although TEVp activity is maximal in salt-free buffers, it is often used in reactions containing 0.2 M NaCl (Eisenmesser et al, 2000;Nallamsetty et al, 2004), and well tolerates NaCl (Cabrita et al, 2007) and TEV SH (Van Den Berg et al, 2006) was highly efficient for on-column cleavage on different type of resins (Zhu et al, 2016).…”

Section: Tevp In Vitro Enzymologymentioning

confidence: 99%

Tobacco Etch Virus protease: A shortcut across biotechnologies

Cesaratto

Burrone

Petris

2016

Journal of Biotechnology

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Cleavage of fusion proteins on the affinity resins using the TEV protease variant

Cited by 16 publications

References 32 publications

Investigating the applicability of human annexin A1 as an affinity tag for separation and purification of the target proteins

Investigating the applicability of human annexin A1 as an affinity tag for separation and purification of the target proteins

Isolation of Protein Complexes from Tobacco Leaves by a Two‐Step Tandem Affinity Purification

Tobacco Etch Virus protease: A shortcut across biotechnologies

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