On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains

Carmignotto, Gabriela Pannunzio; Azzoni, Adriano Rodrigues

doi:10.1016/j.jbiotec.2019.09.012

Cited by 31 publications

(29 citation statements)

References 53 publications

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“…The producer strain obtaining and optimization of cultivation conditions. For the expression of sso7d-Taq in E. coli, the BL21 (DE3) strain was chosen, which is characterized by rapid growth on Luria-Bertani medium and a high level of expression of heterologous proteins [17,18]. For maximum production of the recombinant Sso7d-Taq protein, the following conditions were optimized: the density of the bacterial culture at which the T7 promoter was activated, the induction temperature, the concentration of the IPTG induction activator, and the incubation time of the recombinant culture with IPTG.…”

Section: Fig 3 Pcr Screening Of Taq-pol Clonesmentioning

confidence: 99%

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Sarsen¹,

Akishev²,

Saginova³

et al. 2021

Eurasian Journal of Applied Biotechnology

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Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

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Section: Fig 3 Pcr Screening Of Taq-pol Clonesmentioning

confidence: 99%

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Sarsen¹,

Akishev²,

Saginova³

et al. 2021

Eurasian Journal of Applied Biotechnology

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“…During translation, the available rare tRNA in low amount can diminish the quality and quantification of the protein yield. The excess of these codons even leads to translational troubles (Carmignotto & Azzoni, 2019;Kane, 1995). It has been stated that Cas9 protein has 137 rare codons in the gene sequence even if the heterologous expression in E. coli is stated to be available (Carmignotto & Azzoni, 2019).…”

Section: 4mentioning

confidence: 99%

“…Since mRNA is not highly stable as DNA, the usage of Cas9 mRNA and sgRNA has been provided transient expression with low offtarget effects. However, delivery of Cas9 mRNA in genome editing studies showed no high efficiency (Carmignotto & Azzoni, 2019;Chu et al, 2016;Li et al, 2018). As another delivery method, the ribonucleoprotein (RNP) complexes containing in vitro transcribed gRNA and recombinant Cas9 protein has been demonstrated promising results in decrease offtarget effect (S. Kim et al, 2014;S.…”

Section: Introductionmentioning

confidence: 99%

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The Development of Recombinant hSpCas9 Production System in E. coli

Uslu¹,

Serasker²,

Kocabaş³

2021

genediting

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“…Notably, many affinity tags are available commercially that meet several, if not many, of those requirements. We analyzed two proteins and six peptide affinity tags, which met all of the above criteria, to purify recombinant proteins expressed in E. coli for their effectiveness [ 157 ]. We studied a subset of the capacity of such tags to purify proteins from three samples of eukaryotic cells.…”

Section: Purificationmentioning

confidence: 99%

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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On the expression of recombinant Cas9 protein in E. coli BL21(DE3) and BL21(DE3) Rosetta strains

Cited by 31 publications

References 53 publications

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Modification Of Taq DNA Polymerase with Improved Elongation Ability

The Development of Recombinant hSpCas9 Production System in E. coli

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

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