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Generation of a vector suite for protein solubility screening.

Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P - Front Microbiol (2014)

Bottom Line: Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer.Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP.We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Protein Unit, Institut Pasteur de Montevideo Montevideo, Uruguay.

ABSTRACT
Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins have been produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins (RP) with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer. Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP. We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

No MeSH data available.


Related in: MedlinePlus

Protein production screening in the vector suite. Panels (A,B) corresponds to the E-PAGE 96 acrylamide gels for the expression screening of GFP and DprE1, respectively. The incubation with TEV protease for fusion cleavage is indicated with a +sign over the corresponding lines. Cleaved target protein at the expected molecular weight (MW) is depicted. Additionally, induction temperatures are indicated over each panel. (C) Expression screening for MPK4 at 17°C using a Labchip GX II (Caliper, USA) microfluidic detection system. Arrows indicate the presence of a band with the expected molecular weight. Construct names are provided over each gel line. Solubility improvement with vector suite is indicated by arrows. (D) Analytical size exclusion chromatography (SEC) of the IMAC purified fraction of DsbC-MPK4. Peaks at 7.6 and 8.4 ml correspond to the exclusion volume and the 600 kDa decameric form of DsbC-MPK4, respectively. The 12% SDS-PAGE shows the fusion protein obtained by IMAC purification and DsbC-MPK4 digested by TEV protease. The expected molecular weight of MPK4 (41.7 kDa) is indicated by an arrow.
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Figure 2: Protein production screening in the vector suite. Panels (A,B) corresponds to the E-PAGE 96 acrylamide gels for the expression screening of GFP and DprE1, respectively. The incubation with TEV protease for fusion cleavage is indicated with a +sign over the corresponding lines. Cleaved target protein at the expected molecular weight (MW) is depicted. Additionally, induction temperatures are indicated over each panel. (C) Expression screening for MPK4 at 17°C using a Labchip GX II (Caliper, USA) microfluidic detection system. Arrows indicate the presence of a band with the expected molecular weight. Construct names are provided over each gel line. Solubility improvement with vector suite is indicated by arrows. (D) Analytical size exclusion chromatography (SEC) of the IMAC purified fraction of DsbC-MPK4. Peaks at 7.6 and 8.4 ml correspond to the exclusion volume and the 600 kDa decameric form of DsbC-MPK4, respectively. The 12% SDS-PAGE shows the fusion protein obtained by IMAC purification and DsbC-MPK4 digested by TEV protease. The expected molecular weight of MPK4 (41.7 kDa) is indicated by an arrow.

Mentions: In order to evaluate the expression capabilities and functionality of this new vector suite we selected green fluorescent protein (GFP) as control protein and two “difficult to express” RP such as DprE1 and the MAP kinase 4 from Leishmania major (MPK4). All of them were cloned into 12 different vectors and their expression was evaluated. The results showed that all the GFP constructs were produced soluble and at the expected molecular weight. Fractions treated with TEV showed the correct cleavage and release of GFP protein and fusion partner (Figure 2A). The construct DsbC-GFP under the control of T7 promoter was the less productive when working at 37°C. This was over-passed when the expression was done at 17°C over night (ON) where an increment of cleaved proteins was obtained in most of the cases (Figure 2A).


Generation of a vector suite for protein solubility screening.

Correa A, Ortega C, Obal G, Alzari P, Vincentelli R, Oppezzo P - Front Microbiol (2014)

Protein production screening in the vector suite. Panels (A,B) corresponds to the E-PAGE 96 acrylamide gels for the expression screening of GFP and DprE1, respectively. The incubation with TEV protease for fusion cleavage is indicated with a +sign over the corresponding lines. Cleaved target protein at the expected molecular weight (MW) is depicted. Additionally, induction temperatures are indicated over each panel. (C) Expression screening for MPK4 at 17°C using a Labchip GX II (Caliper, USA) microfluidic detection system. Arrows indicate the presence of a band with the expected molecular weight. Construct names are provided over each gel line. Solubility improvement with vector suite is indicated by arrows. (D) Analytical size exclusion chromatography (SEC) of the IMAC purified fraction of DsbC-MPK4. Peaks at 7.6 and 8.4 ml correspond to the exclusion volume and the 600 kDa decameric form of DsbC-MPK4, respectively. The 12% SDS-PAGE shows the fusion protein obtained by IMAC purification and DsbC-MPK4 digested by TEV protease. The expected molecular weight of MPK4 (41.7 kDa) is indicated by an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3934309&req=5

Figure 2: Protein production screening in the vector suite. Panels (A,B) corresponds to the E-PAGE 96 acrylamide gels for the expression screening of GFP and DprE1, respectively. The incubation with TEV protease for fusion cleavage is indicated with a +sign over the corresponding lines. Cleaved target protein at the expected molecular weight (MW) is depicted. Additionally, induction temperatures are indicated over each panel. (C) Expression screening for MPK4 at 17°C using a Labchip GX II (Caliper, USA) microfluidic detection system. Arrows indicate the presence of a band with the expected molecular weight. Construct names are provided over each gel line. Solubility improvement with vector suite is indicated by arrows. (D) Analytical size exclusion chromatography (SEC) of the IMAC purified fraction of DsbC-MPK4. Peaks at 7.6 and 8.4 ml correspond to the exclusion volume and the 600 kDa decameric form of DsbC-MPK4, respectively. The 12% SDS-PAGE shows the fusion protein obtained by IMAC purification and DsbC-MPK4 digested by TEV protease. The expected molecular weight of MPK4 (41.7 kDa) is indicated by an arrow.
Mentions: In order to evaluate the expression capabilities and functionality of this new vector suite we selected green fluorescent protein (GFP) as control protein and two “difficult to express” RP such as DprE1 and the MAP kinase 4 from Leishmania major (MPK4). All of them were cloned into 12 different vectors and their expression was evaluated. The results showed that all the GFP constructs were produced soluble and at the expected molecular weight. Fractions treated with TEV showed the correct cleavage and release of GFP protein and fusion partner (Figure 2A). The construct DsbC-GFP under the control of T7 promoter was the less productive when working at 37°C. This was over-passed when the expression was done at 17°C over night (ON) where an increment of cleaved proteins was obtained in most of the cases (Figure 2A).

Bottom Line: Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer.Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP.We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

View Article: PubMed Central - PubMed

Affiliation: Recombinant Protein Unit, Institut Pasteur de Montevideo Montevideo, Uruguay.

ABSTRACT
Recombinant protein expression has become an invaluable tool for academic and biotechnological projects. With the use of high-throughput screening technologies for soluble protein production, uncountable target proteins have been produced in a soluble and homogeneous state enabling the realization of further studies. Evaluation of hundreds conditions requires the use of high-throughput cloning and screening methods. Here we describe a new versatile vector suite dedicated to the expression improvement of recombinant proteins (RP) with solubility problems. This vector suite allows the parallel cloning of the same PCR product into the 12 different expression vectors evaluating protein expression under different promoter strength, different fusion tags as well as different solubility enhancer proteins. Additionally, we propose the use of a new fusion protein which appears to be a useful solubility enhancer. Above all we propose in this work an economic and useful vector suite to fast track the solubility of different RP. We also propose a new solubility enhancer protein that can be included in the evaluation of the expression of RP that are insoluble in classical expression conditions.

No MeSH data available.


Related in: MedlinePlus