Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

Santos, Bryan D; Morones‐Ramírez, José Rubén; Balderas-Renterı́a, Isaı́as; Casillas-Vega, Néstor; Zárate, Xristo

doi:10.1007/s12033-019-00176-4

Cited by 14 publications

(15 citation statements)

References 37 publications

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“…A protein band, corresponding to the expected position of SmbP‐hGH (approximately 32 kDa), was observed only in the hypotonic fractions. Previous experiments performed in our laboratory showed that red fluorescent protein tagged with PelB‐SmbP was mostly present in that same fraction 13. Interestingly, when the process was upscaled to a larger volume, some SmbP‐hGH was also observed in the hypertonic fraction.…”

Section: Resultsmentioning

confidence: 74%

“…Cells from the 1‐L culture were harvested by centrifugation at 4 °C, and SmbP‐hGH was extracted from the periplasm by osmotic shock 13. The cells were resuspended in a hypertonic solution (200 m m Tris/HCl, 20% sucrose, 2 m m EDTA, 0.5 mg·mL −1 lysozyme, pH 8.0) and incubated for 1 h at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Experiments in our laboratory have shown that SmbP and CusF function as solubility tags and can be purified employing IMAC, obtaining high purity, and very importantly, excellent final yields since these proteins have a molecular weight of only 10 kDa. We have made improvements to both proteins to increase protein quantity and purity [12,13]. In the case of SmbP, its signal peptide was exchanged for three different sequences: the signal peptides from the proteins CusF, PelB (Erwinia carotovora), and TorA (E. coli).…”

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confidence: 99%

“…Using any of the three different signal peptides, the periplasmic expression of fluorescent proteins marked with SmbP increased more than a 1000-fold, PelB-SmbP being slightly better than CusF-SmbP. As for TorA-SmbP, it transported folded proteins to the cell periplasm with a little less efficiency; although protein production with any of the different signal sequences is target protein-dependent [13].…”

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confidence: 99%

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Engineered small metal‐binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli

et al. 2020

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Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.

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Section: Resultsmentioning

confidence: 74%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Engineered small metal‐binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli

et al. 2020

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“…The columns MBP, Talon, Ni–NTA, and GST were the least costly, with resins costing $12–36 to purify the labeled polypeptide by 10 mg. To purify 10 mg of marked polypeptide, calmodulin (for CBP tag) and Strep-Tactin (for Strep II tag) were notably costlier at $114–293. The FLAG and HPC monoclonal resins are especially costly at $1000–5000 for the same volume of marked polypeptide due to their large unit resin cost and relatively small capacity (0.6 mg FLAG marked protein/mL resin versus 5–10 mg HIS tagged protein/mL resin) [ 115 , 160 , 161 , 162 ]. Choosing an attraction suffix specifically depends upon the criteria of the experiment.…”

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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Expression and Purification of Recombinant Proteins in Escherichia coli Tagged with the Metal-Binding Proteins SmbP and CusF3H+

Gómez‐Loredo

Santos

Perez-Perez

et al. 2020

Methods in Molecular Biology

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Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

Cited by 14 publications

References 37 publications

Engineered small metal‐binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli

Engineered small metal‐binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli

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

Expression and Purification of Recombinant Proteins in Escherichia coli Tagged with the Metal-Binding Proteins SmbP and CusF3H+

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