Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature

San-Miguel, Teresa; Pérez‐Bermúdez, Pedro; Gavidia, Isabel

doi:10.1186/2193-1801-2-89

Cited by 89 publications

(44 citation statements)

References 19 publications

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“…The pDEST15-TSN2 construct was transformed into Escherichia coli BL21 (DE3) RIL (Stratagene) cells. Induction of the expression of the recombinant protein was performed as described previously (San-Miguel et al, 2013). Briefly, protein expression was induced at OD 600 = 0.5 by adding 1 mM isopropyl b-D-1-thiogalactopyranoside to Luria-Bertani medium supplemented with 100 mg/mL ampicillin and 2 g/L glucose.…”

Section: Preparation Of Antibodiesmentioning

confidence: 99%

Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis

et al. 2015

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Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress.

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Section: Preparation Of Antibodiesmentioning

confidence: 99%

Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis

et al. 2015

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“…The classic problem of inclusion body aggregation in overexpression in E. coli [31] was overcome by a combination of low temperature incubation as shown for TPases of transposons such as Ac, Tol2, Mos1 and Mboumar-9 [25,[33][34][35], and early log-phase induction as previously described [32]. In our study, the percentage of expressed soluble protein increased with a concomitant reduction of endogenous host proteins making for a more efficient purification process.…”

Section: Discussionmentioning

confidence: 50%

Prokaryotic Expression and Purification of Soluble Goldfish Tgf2 Transposase with Transposition Activity

Shen

Hou

et al. 2014

Mol Biotechnol

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Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.

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“…One of the obstacles for obtaining large amounts of recombinant proteins in E. coli is the inclusion bodies (De Marco, 2009). It is known that altering the growth conditions can affect soluble protein expression level by varying the folding environments of the recombinant protein, such as initial culture density, temperature, and duration of the expression stage (San-Miguel et al, 2013). Besides, the growth and induction of cells under heat-shock, osmotic stress, and osmole supplementation conditions have been shown to enhance solubility of some recombinant proteins (Harrison and Bagajewicz, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Escherichia coli is widely used for expressing recombinant proteins; however, there are still bottlenecks for obtaining large amounts of soluble and functional proteins (San-Miguel et al, 2013). Variation in environmental condition was reported to influence the recombinant protein production (Hoffmann and Rinas, 2004;Jamal et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Omics Analysis Reveals the Mechanism of Enhanced Recombinant Protein Production Under Simulated Microgravity

Huangfu

Kim

et al. 2020

Front. Bioeng. Biotechnol.

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Simulated microgravity (SMG) is regarded as a suitable environment to produce recombinant proteins. This study showed that β-glucuronidase expressing Escherichia coli had higher productivity of recombinant protein and higher plasmid copy number under SMG compared with the normal gravity condition. The cellular changes were analyzed at both transcriptomic and proteomic levels. The upregulation of a group of ribosome/RNA polymerase genes and a cluster of genes involving energy metabolism at transcriptomic level stood out for improved production of recombinant protein under SMG. The protein folding modulators such as chaperones were upregulated at proteomic level, which could be a result of the increased activity of protein synthesis and can help recombinant protein production. Protein export was also strengthened, which was revealed at both transcriptomic and proteomic levels. The results demonstrated that SMG is a favorable environment for recombinant protein production arousing the upregulation of protein synthesis, protein folding, and protein export.

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Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature

Cited by 89 publications

References 19 publications

Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis

Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis

Prokaryotic Expression and Purification of Soluble Goldfish Tgf2 Transposase with Transposition Activity

Omics Analysis Reveals the Mechanism of Enhanced Recombinant Protein Production Under Simulated Microgravity

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