A pre-packed tandem affinity chromatography column

Abstract

The present invention discloses a pre-loaded tandem affinity chromatography column, including a column tube, wherein the upper and lower parts of the column tube are respectively provided with an upper sealing cover and a lower sealing cover, the upper part of the upper sealing cover is provided with a liquid inlet, the bottom of the lower sealing cover is provided with a liquid outlet, and the column tube is provided with a plurality of sieve plates from top to bottom, and an affinity medium is filled between two adjacent sieve plates. A bottom sieve plate is provided at the bottom of the column tube, and an affinity medium 2 is filled in the upper part of the bottom sieve plate, and a middle sieve plate is provided above the affinity medium 2, and an affinity medium 1 is filled in the upper part of the middle sieve plate, and an upper sieve plate is provided above the affinity medium 1, and the two affinity media have a small cross reaction, and the two affinity media are not connected in space. The beneficial effects of the present invention are: the affinity chromatography column can simplify the purification and labeling steps, and does not require steps such as gradient elution and desalting, so that purification and labeling can be completed on the same column, and the operation is simple, fast and convenient.

Description

Technical Field

[0001] The invention relates to the technical field of chromatography columns, in particular to a pre-packed tandem affinity chromatography column. Background Art

[0002] Protein expression and purification is a common experimental technique in biological research and industrial medicine production. It uses the molecular weight, hydrophobicity, hydrophilicity and other properties of the protein to separate the target protein from the protein mixture through various corresponding chromatography columns and other methods. Among them, affinity chromatography has become the preferred method for general protein purification because of its specificity and high yield. However, affinity chromatography requires a label to be carried on the target protein.

[0003] In order to remove the tag, a common method is to add a specific protease cleavage site between the tag and the protein, purify with a specific tag, and then add a specific enzyme containing the same tag for cleavage. After the reaction is completed, the flow-through is collected by affinity chromatography using the tag to obtain the target protein solution. This process requires two purifications, and the protein purified for the first time needs to be desalted. On-column enzyme cleavage can avoid multiple tedious steps, but the use of enzymes with the same tag will cause incomplete spatial crossover between the enzyme and the substrate, affecting the reaction. If a non-tagged enzyme is used, the product will require secondary purification. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the above technical defects and provide a pre-loaded tandem affinity chromatography column, which can simplify the purification and labeling steps, does not require steps such as gradient elution and desalting, and allows purification and labeling to be completed on the same column.

[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a pre-packed tandem affinity chromatography column, including a column tube, the upper and lower parts of the column tube are respectively provided with an upper sealing cover and a lower sealing cover, the upper part of the upper sealing cover is provided with a liquid inlet, and the bottom of the lower sealing cover is provided with a liquid outlet, and several layers of sieve plates are provided inside the column tube from top to bottom, and affinity medium is filled between two adjacent sieve plates.

[0006] Furthermore, the sieve plate is tightly combined with the tube wall, and the affinity medium is also tightly combined with the sieve plate without leaving any gap.

[0007] Furthermore, a bottom sieve plate is provided at the bottom of the column tube, affinity medium 2 is filled on the upper part of the bottom sieve plate, a middle sieve plate is provided above affinity medium 2, affinity medium 1 is filled on the upper part of the middle sieve plate, and an upper sieve plate is provided above affinity medium 1. The two affinity media have a small cross-reaction, and the two affinity media are not connected in space.

[0008] Furthermore, the affinity medium 1 and the affinity medium 2 respectively use two labels of different sizes as the medium.

[0009] Furthermore, of the two affinity media, affinity media 1 located at the upper portion binds more proteins per unit volume, and the medium that binds more proteins per unit volume is in the upper layer, and the target protein obtains a higher concentration.

[0010] Furthermore, the upper sealing cover and the lower sealing cover are both detachable screw covers for connection with the pressure system.

[0011] Furthermore, the method for making a pre-packed tandem affinity chromatography column is as follows: prepare an empty column tube; tightly embed a lower sieve plate of the same size as the inner bottom surface of the empty column outlet into the inner bottom surface, fill the sieve plate with 0.5 ml of affinity medium 2, seal the top of affinity medium 2 with the same sieve plate without leaving obvious gaps, the sieve plate is tightly combined with the tube wall, fill 1-2 ml of affinity medium 1 above the sieve plate, and seal it again with a sieve plate.

[0012] The beneficial effects of the present invention are as follows: the affinity chromatography column can simplify the purification and labeling steps, does not require steps such as gradient elution and desalting, and allows purification and labeling to be completed on the same column, which is simple, fast and convenient to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic structural diagram of a pre-packed tandem affinity chromatography column of the present invention.

[0014] As shown in the figure: 11, liquid inlet, 12, upper sealing cover, 21, column tube, 22, upper sieve plate, 23, affinity medium 1, 24, middle sieve plate, 25, affinity medium 2, 26, bottom sieve plate, 31, lower sealing cover, 32, liquid outlet. DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] In conjunction with the accompanying drawings, a pre-installed tandem affinity chromatography column includes a column tube 21, wherein the upper and lower parts of the column tube 21 are respectively provided with an upper sealing cover 12 and a lower sealing cover 31, wherein the upper sealing cover 12 is provided with a liquid inlet 11, and the lower sealing cover 31 is provided with a liquid outlet 32 ​​at the bottom, and the column tube 21 is provided with several layers of sieve plates from top to bottom, and an affinity medium is filled between two adjacent sieve plates. The sieve plate is tightly combined with the tube wall, and the affinity medium is also tightly combined with the sieve plate, leaving no gap visible to the naked eye. The excess gap will cause the protein solution to be mixed and diluted therein, affecting the purification effect. A bottom sieve plate 26 is provided at the bottom of the column tube 21, and an affinity medium 25 is filled on the top of the bottom sieve plate 26, and a middle sieve plate 24 is provided above the affinity medium 25, and an affinity medium 1 23 is filled on the top of the middle sieve plate 24, and an upper sieve plate 22 is provided above the affinity medium 1 23, and the two affinity media have a small cross reaction, and the two affinity media are not connected in space. The sieve plate below prevents leakage of the lower medium, the middle sieve plate 24 prevents the two media from mixing, and the upper sieve plate 22 filters the particles in the sample protein mixture. The affinity medium 1 23 and the affinity medium 2 25 are respectively used as media with two labels of different sizes. The use of small labels may cause the problem of low binding efficiency caused by the position. The use of large labels will cause the fusion protein to be too large and unfavorable for expression and purification. Among the two affinity media, the medium unit volume of the affinity medium 1 23 located at the top is combined with more proteins, and the medium unit volume is combined with more proteins in the upper layer, and the target protein obtains a higher concentration. The upper medium is initially combined with the target protein, and the lower medium is combined with the enzyme and the trace amount of the label protein that falls off after enzyme cutting, such as the uncut or cut label part. The upper sealing cover 12 and the lower sealing cover 31 are both detachable screw caps for connecting to the pressure system.

[0017] The prepacked column provided by the present invention is used for the purification and de-labeling of a fusion protein of a small tag-large tag-TEV site-target protein. A mixed solution containing the target protein enters from a liquid inlet 11, the target protein is bound to an upper medium, eluted using a lower eluent, and after balancing with a TEV enzyme buffer, the tag-TEV enzyme bound to the lower layer is added for enzymatic cleavage, and the flow-through is the target protein.

[0018] This specific example demonstrates the implementation and use of the technical solution using a Ni ion affinity-Amylose tandem mini-column used to purify the His6-MBP-TEV-target protein and remove its tag.

[0019] In this specific embodiment, a screw-type hollow column with detachable upper and lower liquid inlet and outlet ports 32 is used, and an integrated gravity column hollow column may also be used. The sieve plate is a sieve plate commonly used in protein chromatography columns, which can pass the solution but not the medium particles.

[0020] When the empty column is ready, the lower sieve plate of the same size as the inner bottom surface of the empty column liquid outlet 32 ​​is closely embedded in the inner bottom surface. The sieve plate is filled with 0.5 ml of Amylose medium (about 6 mg of MBP fusion protein can be bound per 1 ml of medium), and the Amylose medium is sealed with the same sieve plate above, and no obvious gap is left, and the sieve plate is tightly combined with the tube wall. 1-2 ml of Ni-NTA (other types of Ni ion media can also be used) is filled above the sieve plate, and the sieve plate is sealed again on it to filter the particles that cannot be removed in the initial protein mixture and reduce the impact of sample loading on the medium scouring.

[0021] Before purification, the column was balanced with binding buffer. The protein mixture containing His6-MBP-tev-target protein was passed through the column, and most of the His6-MBP-tev-target protein was bound to the upper Ni-NTA. Then 6 column volumes of binding buffer were used to wash away non-specifically bound proteins, and the binding buffer containing an appropriate concentration of maltose was used to elute the proteins bound to Amylose. Tev enzyme reaction buffer was added to the column, and 0.5 ml of the upper filler volume of MBP-Tev enzyme (containing 1 mg) was added, and incubated at 30 ° C for 3 h or 4 ° C overnight. The excised protein was eluted using an elution buffer without imidazole and maltose, and 2-3 ml of volume could be collected in steps for scanning and gel running.

[0022] The present invention and its implementation methods are described above, which is not restrictive and is only one of the implementation methods of the present invention. The actual implementation methods are not limited thereto. In short, if ordinary technicians in the field are inspired by it and design structural methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A prepacked tandem affinity chromatography column, comprising a column tube, characterized in that: The upper and lower parts of the column tube are respectively provided with an upper sealing cover and a lower sealing cover, the upper part of the upper sealing cover is provided with a liquid inlet, and the bottom of the lower sealing cover is provided with a liquid outlet. The column tube is provided with several layers of sieve plates from top to bottom, and affinity medium is filled between two adjacent sieve plates. The sieve plate is tightly combined with the tube wall, and the affinity medium is also tightly combined with the sieve plate without leaving any gaps; A bottom sieve plate is provided at the bottom of the column tube, affinity medium 2 is filled on the upper part of the bottom sieve plate, a middle sieve plate is provided above affinity medium 2, affinity medium 1 is filled on the upper part of the middle sieve plate, and an upper sieve plate is provided above affinity medium 1; The affinity medium 1 and the affinity medium 2 respectively use two labels of different sizes as the medium; of the two affinity media, the affinity medium 1 located at the upper part binds more proteins per unit volume, and the target protein obtains a higher concentration; the upper sealing cover and the lower sealing cover both use detachable screw caps for connecting to the pressure system.

2. A prepacked tandem affinity chromatography column according to claim 1, characterized in that: The method for making a pre-packed tandem affinity chromatography column is as follows: prepare an empty column tube; tightly embed a lower sieve plate of the same size as the inner bottom surface of the empty column outlet into the inner bottom surface, fill the sieve plate with 0.5 ml of affinity medium 2, seal the top of affinity medium 2 with the same sieve plate without leaving obvious gaps, tightly combine the sieve plate with the tube wall, fill 1-2 ml of affinity medium 1 above the sieve plate, and seal it again with a sieve plate to obtain the pre-packed tandem affinity chromatography column.

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