DE202023104491U1 - A system for active protein purification with high yield - Google Patents

Abstract

A system for active protein purification with high yield, consisting of:
a first purification unit for purification of the protein using nickel column chromatography (Ni-NTA) and for treatment with mild urea and 0.15 mM DDM (surfactant);
a second purification unit for performing ion exchange chromatography on treated protein; and
a third purification unit for performing size exclusion chromatography on proteins purified by ion exchange chromatography to obtain a purified protein.

Description

FIELD OF THE INVENTION

The present disclosure relates to the field of pharmaceutical and computational biology. In particular, the present disclosure relates to a high yield active protein purification system wherein two active proteins, namely adipoR1 and adipoR2, are successfully purified.

BACKGROUND OF THE INVENTION

Adiponectin, an adipokine involved in the control of obesity and diabetes, is bound by the membrane proteins AdipoR1 and AdipoR2. When the ligand adiponectin binds to the AdipoR1/R2 receptor, the signaling pathway is initiated. Then the signals for fatty acid oxidation, glucose uptake and other cellular processes are activated. The sophisticated in vitro protein purification techniques for the receptors typically provide modest yields. Therefore, there is a need for a purification approach that enables a high yield in the purification of such proteins, whereby a high yield of such proteins may contribute to the development of effective therapeutics against obesity and diabetes.

From the perspective of the previous discussion, it is clear that there is a need for a high-yield active protein purification system in which two active proteins, namely adipoR1 and adipoR2, are successfully purified.

SUMMARY OF THE INVENTION

The present disclosure relates to a high yield active protein purification system wherein two active proteins, namely adipoR1 and adipoR2, are successfully purified. In the present invention, the mammalian protein adipoR1 is expressed in the pETDuet-1 vector as part of the new purification technique. Ion exchange chromatography and a TED nickel column are used for standard purification. There were still some impurities present in the protein. The new technique uses a Ni-NTA column, 0.15 mM DDM (surfactant), and mild urea treatment, followed by ion exchange and size exclusion chromatography. The addition of this new step helped to obtain pure protein. Protein activity was assessed using biolayer interferometry and computer analysis. The binding activity of our protein was successfully demonstrated. The protein yield of the culture is 10 mg/L.

The present disclosure aims to provide a system for purifying active proteins with high yield.

An aim of the present disclosure is to provide a high yield active protein purification system wherein two active proteins, namely adipoR1 and adipoR2, are successfully purified

Another aim of the present disclosure is to purify the active adipoR2 protein.

Another object of the present disclosure is to provide a system for purification that provides high yield and enables NMR and X-ray crystallography to be performed using purified proteins.

Another aim of the present disclosure is to assess protein activity using biolayer interferometry and computational studies

Another aim of the present disclosure is to perform molecular docking studies of the purified proteins.

In order to further illustrate the advantages and features of the present disclosure, a more detailed description of the invention will be made with reference to specific embodiments thereof shown in the accompanying drawings. It is understood that these drawings represent only typical embodiments of the invention and are therefore not to be viewed as limiting its scope. The invention is described and explained in more detail and in greater detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

• 1 veranschaulicht ein Blockdiagramm eines Systems zur aktiven Proteinreinigung mit hoher Ausbeute gemäß einer Ausführungsform der vorliegenden Offenbarung; Und
• 2 zeigt ein Diagramm, das die Ergebnisse von BLITZ für Adiponecting und AdipoRI /R2 mit Ni-Sensoren gemäß einer Ausführungsform der vorliegenden Offenbarung zeigt.

These and other features, aspects and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like reference numerals represent like parts throughout the drawings, in which:

• 1 illustrates a block diagram of a system for active, high-yield protein purification in accordance with an embodiment of the present disclosure; And
• 2 Figure 3 is a graph showing BLITZ results for Adiponecting and AdipoRI/R2 with Ni sensors according to an embodiment of the present disclosure.

Additionally, experienced craftsmen will recognize that elements in the drawings are presented for convenience and may not necessarily have been drawn to scale. For example, the flowcharts illustrate the method through key steps that help improve understanding of aspects of the present disclosure. Additionally, in view of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only the specific details relevant to understanding the embodiments of the present disclosure around the drawings not to be obscured by details that would be readily apparent to one of ordinary skill in the art who would benefit from the description herein.

DETAILED DESCRIPTION:

In order to promote understanding of the principles of the invention, reference will now be made to the embodiment shown in the drawings and specific language will be used to describe the same. It is to be understood, however, that this is not intended to limit the scope of the invention, since changes and further modifications to the system illustrated and further applications of the principles of the invention set forth therein are contemplated as would normally occur to one skilled in the art Technology to which the invention relates.

Those skilled in the art will understand that the foregoing general description and the following detailed description are exemplary and illustrative of the invention and are not intended to limit the same.

References in this specification to “an aspect,” “another aspect,” or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure is included. Therefore, the phrases “in one embodiment,” “in another embodiment,” and similar phrases in this specification may, but not necessarily, refer to the same embodiment.

The terms "includes", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, such that a process or method that includes a list of steps not only includes those steps, but may include other steps not expressly listed or are inherent in that process or method. Likewise, one or more devices or subsystems or elements or structures or components prefixed with "comprises...a" do not exclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures from other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which this invention pertains. The system, methods and examples provided herein are for illustrative purposes only and are not intended to be limiting.

Embodiments of the present disclosure are described in detail below with reference to the accompanying drawings.

1 shows a block diagram of a system for active, high-yield protein purification according to an embodiment of the present disclosure. The system (100) comprises a first purification unit (102) for purifying the protein using nickel column chromatography (Ni-NTA) and for treatment with mild urea and 0.15 mM DDM (surfactant).

In one embodiment, a second purification unit (104) is used to perform ion exchange chromatography on treated protein.

In one embodiment, a third purification unit (106) is used to perform size exclusion chromatography on protein purified by ion exchange chromatography to obtain a purified protein.

In one embodiment, the mammalian proteins, namely adipoR1 and adipoR2, are purified, with the mammalian protein adipoR1 being present in the pETDuet-1 vector.

In one embodiment, Ni-NTA column chromatography is performed using a standard protocol along with minor modifications: a cell lysis buffer is prepared with 50 mM Tris, 1 M NaCl, 10 mM MgCl2, 5 mM ATP, 10% glycerol, 100 mM ammonium sulfate, 100 mM KCl, 2 mM BME and 1 mM PMSF, where the pH is 8.0 and where 50 mM Tris can be divided into 20 mM HEPES buffer can be replaced. 7 g of pellet is suspended in 28 ml of cell lysis buffer, and then lysozyme is added to the solution, and then the suspension is incubated on ice for one hour, and after incubation, ultrasonication is performed for 3 minutes, with centrifugation performed for 45 minutes , then the supernatant is removed and passed over Ni resin (TED resin), followed by washing and finally the protein is eluted with lysis buffer containing 250 mM imidazole.

In one embodiment, the unbound fraction from ion exchange chromatography is subjected to a urea wash with 0.15 mM DDM in a Ni-NTA column using lysis buffer without PMSF, Amm. Sulfate and ATP are used to equilibrate the column. The unbound fraction is run over the Ni-NTA column, washing the column with 0.15 M DDM with urea, washing the column with 10 column volumes of lysis buffer, followed by elution with imidazole buffer, and dialyzing and ionizing eluted protein becomes. Exchange chromatography is carried out.

In one embodiment, the collected protein is run through a gel filtration column and analysis is performed by Western blot, the binding activity of the protein is confirmed using the BLITZ (biolayer interferometry) instrument, and molecular docking is performed to visualize the key interactions involved .

In the present disclosure, the mammalian protein adipoR1 is expressed in the pETDuet-1 vector as part of the new purification technique. Ion exchange chromatography and a TED nickel column are used for standard purification. There were still some impurities present in the protein. Using the new technique for purification, a Ni-NTA column, 0.15 mM DDM (surfactant), a moderate urea treatment and size exclusion and ion exchange chromatography. The addition of this new step helped to obtain pure protein. Protein activity was assessed using biolayer interferometry and computer analysis. The binding activity of our protein was successfully demonstrated. The protein yield of the culture is 10 mg/L.

1. 1. Die Ni-NTA-Säule wird mit dem Standardprotokoll mit folgenden Modifikationen betrieben:
   1. a. Zelllysepuffer : (50 mM Tris , 1 M NaCl, 10 mM MgCl2, 5 mM ATP, 10 % Glycerin, 100 mM Ammoniumsulfat , 100 mM KCl , 2 mM BME, 1 mM PMSF, pH 8.0). Man kann 50 mM ersetzen Tris mit 20 mM HEPES-Puffer.
   2. b. Zelllysepuffer suspendiert . Der Lösung wird Lysozym (Endkonzentration 1 mg/ml) zugesetzt. Die Suspension wird 1 Stunde auf Eis inkubiert.
   3. c. Auf die Inkubation folgt eine 3-minütige Ultraschallbehandlung (Amplitude 25 %, Puls aus 30 Sekunden, Puls ein 30 Sekunden).
   4. d. Die Zentrifugation erfolgt 45 Minuten lang bei 20,000 U/min unter Verwendung eines SS-34-Rotors. Der Überstand wird entnommen und über Ni-Harz (TED-Harz) geleitet und anschließend gewaschen.
   5. e. Im letzten Schritt wird das Protein mit Lysepuffer eluiert , der 250 mM Imidazol (pH 7,4) enthält.
2. 2. Die Ionenaustauschchromatographie wird mit folgenden Spezifikationen durchgeführt: Die QMA-Ionenaustauschsäule wird verwendet, um andere Proteine von adipoR1 zu trennen.
   1. a. Das eluierte Protein wird gegen Puffer mit niedrigem Salzgehalt (50 mM) dialysiert Tris , 350 mM NaCl, 10 mM MgCl2, 10 % Glycerin, 100 mM KCl, 2 mM BME, pH 7.4).
   2. b. Der ungebundene Anteil liegt im Ausschlussvolumen vor. Die Verunreinigung eluiert in einem Puffer mit hohem Salzgehalt (wie ein Puffer mit niedrigem Salzgehalt und 1 M NaCl).
3. 3. Die ungebundene Fraktion wird einer Harnstoffwäsche mit 0.15 mM DDM in einer Ni-NTA-Säule unterzogen.
   1. a. Lysepuffer (ohne PMSF, Ammoniumsulfat und ATP) verwendet.
   2. b. Die ungebundene Fraktion wird über die Ni-NTA-Säule laufen gelassen. Die Säule wird mit 0.15 M DDM mit Harnstoff (100 mM Harnstoff ohne NaCl im Lysepuffer) gewaschen.
   3. c. Die Säule wird mit 10 Säulenvolumina Lysepuffer gewaschen und anschließend mit Imidazolpuffer eluiert.
   4. d. Das eluierte Protein wird dialysiert und die Ionenaustauschchromatographie wird wie in Schritt (2) durchgeführt.
   5. e. Das gesammelte Protein wird über eine Gelfiltrationssäule laufen gelassen und die Analyse erfolgt mittels Western Blot.
   6. f. Die Bindungsaktivität des Proteins wird mit dem BLITZ-Instrument ( Biolayer Interferometry) bestätigt.
   7. g. Zur Visualisierung der wichtigsten beteiligten Wechselwirkungen wird molekulares Docking durchgeführt.

In one embodiment, the new protein purification technique provides high yields and active protein. The proteins can be used for NMR and X-ray crystallography. NMR is very difficult at low yields. However, in the proposed cleaning technique, the number of steps is higher and can be reduced. The proposed purification technique successfully purified adipoR1/R2 receptors, which technique or method involves the use of a Ni-NTA column and mild urea treatment and 0.15 mM DDM (surfactant) followed by ion exchange and size exclusion chromatography. The detailed steps of the proposed cleaning technique are described below.

1. 1. The Ni-NTA column is operated using the standard protocol with the following modifications:
   1. a. Cell lysis buffer: (50 mM Tris, 1 M NaCl, 10 mM MgCl2, 5 mM ATP, 10% glycerol, 100 mM ammonium sulfate, 100 mM KCl, 2 mM BME, 1 mM PMSF, pH 8.0). One can replace 50 mM Tris with 20 mM HEPES buffer.
   2. b. Cell lysis buffer suspended. Lysozyme (final concentration 1 mg/ml) is added to the solution. The suspension is incubated on ice for 1 hour.
   3. c. Incubation is followed by 3 minutes of ultrasound treatment (amplitude 25%, pulse off 30 seconds, pulse on 30 seconds).
   4. d. Centrifugation is performed at 20,000 rpm for 45 minutes using an SS-34 rotor. The supernatant is removed and passed over Ni resin (TED resin) and then washed.
   5. e. In the final step, the protein is eluted with lysis buffer containing 250 mM imidazole (pH 7.4).
2. 2. Ion exchange chromatography is performed with the following specifications: The QMA ion exchange column is used to separate other proteins from adipoR1.
   1. a. The eluted protein is dialyzed against low salt buffer (50 mM Tris, 350 mM NaCl, 10 mM MgCl2, 10% glycerol, 100 mM KCl, 2 mM BME, pH 7.4).
   2. b. The unbound portion is present in the exclusion volume. The contaminant elutes in a high salt buffer (such as a low salt buffer and 1M NaCl).
3. 3. The unbound fraction is subjected to a urea wash with 0.15 mM DDM in a Ni-NTA column.
   1. a. Lysis buffer (without PMSF, ammonium sulfate and ATP) was used.
   2. b. The unbound fraction is run over the Ni-NTA column. The column is washed with 0.15 M DDM with urea (100 mM urea without NaCl in the lysis buffer).
   3. c. The column is washed with 10 column volumes of lysis buffer and then eluted with imidazole buffer.
   4. d. The eluted protein is dialyzed and ion exchange chromatography is performed as in step (2).
   5. e. The collected protein is run through a gel filtration column and analyzed using Western blotting.
   6. f. The binding activity of the protein is confirmed using the BLITZ instrument (Biolayer Interferometry).
   7. G. Molecular docking is performed to visualize the key interactions involved.

2 Figure 3 is a graph showing BLITZ results for Adiponecting and AdipoRI/R2 with Ni sensors according to an embodiment of the present disclosure.

The BLITZ results for adiponectin and AdipoR1/R2 with Ni sensors show the binding of the protein. The molecular docking studies show that hydrophobic residues interact with the key protein residues.

The results show that the present invention can open up new drug development opportunities for the pharmaceutical industry.

The drawings and the description above provide examples of embodiments. Those skilled in the art will recognize that one or more of the elements described can certainly be combined into a single functional element. Alternatively, certain elements can be divided into several functional elements. Elements of one embodiment may be added to another embodiment. For example, the orders of the processes described herein may be changed and are not limited to the manner described herein. Additionally, the actions of a flowchart do not have to be implemented in the order shown; Not all actions necessarily have to be carried out. Even those actions that are not dependent on other actions can be carried out in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples. Numerous variations, whether explicitly stated in the specification or not, such as: B. Differences in structure, dimensions and material use are possible. The scope of the embodiments is at least as broad as indicated by the following claims.

Advantages, other benefits, and solutions to problems have been described above with respect to specific embodiments. However, the advantages, benefits, solutions to problems and any components that may cause a benefit, advantage or solution to occur or become more pronounced should not be construed as a critical, necessary or essential function or component of any or all of the claims.

CREDENTIALS

100

A system for active protein purification with high yield.

102

First cleaning unit

104

Second cleaning unit

106

Third cleaning unit

Claims (9)

A system for active protein purification with high yield, consisting of: a first purification unit for purification of the protein using nickel column chromatography (Ni-NTA) and for treatment with mild urea and 0.15 mM DDM (surfactant); a second purification unit for performing ion exchange chromatography on treated protein; and a third purification unit for performing size exclusion chromatography on proteins purified by ion exchange chromatography to obtain a purified protein. System after Claim 1 , wherein the mammalian proteins, namely adipoR1 and adipoR2, are purified, the mammalian protein adipoR1 being present in the pETDuet-1 vector. System after Claim 1 , where Ni-NTA column chromatography is performed using a standard protocol along with minor modifications. System after Claim 3 , where a cell lysis buffer is prepared with 50 mM Tris, 1 M NaCl, 10 mM MgCl2, 5 mM ATP, 10% glycerol, 100 mM ammonium sulfate, 100 mM KCl, 2 mM BME and 1 mM PMSF, where the pH is 8, 0 and where 50 mM Tris can be replaced with 20 mM HEPES buffer. System after Claim 3 , where 7 g of pellet is suspended in 28 ml of cell lysis buffer, then lysozyme is added to the solution and the suspension is then incubated on ice for one hour and after incubation, ultrasonication is performed for 3 minutes, centrifugation is performed for 45 minutes, and after that, the supernatant is taken and passed over Ni resin (TED resin), followed by washing, and finally the protein is washed with lysis buffer , which contains 250 mM imidazole, elutes. System after Claim 1 , wherein ion exchange chromatography is performed after Ni-NTA column chromatography, where the QMA ion exchange column is used to separate other proteins from AdipoR1, where the protein eluted in Ni-NTA column chromatography is dialyzed against a low salt buffer with a pH of 7.4 and containing 50 mM Tris, 350 mM NaCl, 10 mM MgCl ₂ , 10% glycerol, 100 mM KCl and 2 mM BME, with the unbound fraction emerging in the exclusion volume where the impurity elutes in high salt buffer. System after Claim 1 , whereby the unbound fraction from the ion exchange chromatography is subjected to a urea wash with 0.15 mM DDM in a Ni-NTA column, using lysis buffer without PMSF, Amm. Sulfate and ATP are used to equilibrate the column. System after Claim 7 , wherein the unbound fraction is run over the Ni-NTA column, the column is washed with 0.15 M DDM with urea, the column is washed with 10 column volumes of lysis buffer and then eluted with imidazole buffer, and eluted protein is dialyzed and ion exchange chromatography is carried out. System after Claim 1 , where the collected protein is passed through a gel filtration column and the analysis is performed by western blot, the binding activity of the protein is confirmed with the BLITZ instrument (biolayer interferometry), and molecular docking is performed to visualize the key interactions.

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