Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli

Zou, Zhurong; Cao, Lijuan; Zhou, Pei; Su, Yu-Ching; Sun, Yongjun; Li, Wenjuan

doi:10.1016/j.jbiotec.2008.05.007

Cited by 32 publications

(19 citation statements)

References 23 publications

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“…To tackle this problem, we constructed a bacterial expression vector system harboring 13 different solubility-tags, which allowed us to obtain soluble SAS-5 FL fused to MsyB (Zou et al, 2008) in quantities sufficient for biophysical analysis (Figure 1—figure supplement 1, which shows all recombinant proteins in this study). Characterization of such purified SAS-5 FL using circular dichroism (CD) revealed the presence of protein aggregates (Figure 1—figure supplement 2A,B), a conclusion also supported by size-exclusion chromatography multi-angle light scattering (SEC-MALS, Figure 1—figure supplement 2C) and negative-stain electron microscopy (Figure 1—figure supplement 2D).…”

Section: Resultsmentioning

confidence: 99%

The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

Rogala

Dynes

Hatzopoulos

et al. 2015

eLife

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Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.DOI: http://dx.doi.org/10.7554/eLife.07410.001

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Section: Resultsmentioning

confidence: 99%

The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

Rogala

Dynes

Hatzopoulos

et al. 2015

eLife

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“…In addition, these solubility enhancers might directly act as intramolecular chaperones by participating in native folding of the target proteins [19]. This family of fusion proteins includes the lipoyl domain from B. Stearothermophilus E2p [24], consisting of a short acidic 109 amino acid tag (pI: 4.53 and MW: 11 994.3 Da).…”

Section: Choice Of Fusion Proteinsmentioning

confidence: 99%

Production of prone‐to‐aggregate proteins

Lebendiker¹,

Danieli²

2013

FEBS Letters

128

102

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a b s t r a c tExpression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and costeffective method for producing proteins in basic research and for pharmaceutical applications. Despite accumulating experience and methodologies developed over the years, production of recombinant proteins prone to aggregate in E. coli-based systems poses a major challenge in most research applications. The challenge of manufacturing these proteins for pharmaceutical applications is even greater. This review will discuss effective methods to reduce and even prevent the formation of aggregates in the course of recombinant protein production. We will focus on important steps along the production path, which include cloning, expression, purification, concentration, and storage.

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“…Different approaches were then implemented to solve this problem: from the use of different E. coli strains, such as DH5α, Rosetta (DE 3 ) pLysS, BL21 and Krx (strain Krx allowed higher expression levels) to control of the temperature growth (TEVp is prone to aggregate at higher temperatures) (Fang et al, 2007;Kapust and Waugh, 1999;Kapust et al, 2002aKapust et al, , 2001Miladi et al, 2011;Van Den Berg et al, 2006;Wu et al, 2009). TEVp was also obtained following refolding of the material recovered from inclusion bodies (Blommel and Fox, 2007;Lucast et al, 2001) and through the widely used approach of fusing it to solubility tags such as poly-histidine or polyarginine tags (Kapust and Waugh, 1999;Kapust et al, 2001;Kapust et al, 2002b), Maltosebinding protein (MBP) and glutathione S-transferase (GST) (Blommel and Fox, 2007;Kapust and Waugh, 1999;Sun et al, 2012), Thioredoxin (TRX) (Kapust and Waugh, 1999), Streptag II (Miladi et al, 2011), hyper-acidic protein fusion partners (Zou et al, 2008), SUMO (Zou et al, 2008), the N-utilization substance (NusA) (Zou et al, 2008), superfolded GFP or chaperone proteins (Fang et al, 2007). Combining two or more of these variables it is now possible to obtain high scale production of soluble active and highly pure TEVp of up to 300-400 mg/L from bacterial cultures (Blommel and Fox, 2007;Wu et al, 2009).…”

Section: Solubilitymentioning

confidence: 99%