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A method for large-scale purification of gram-level mechanically functional proteins

A large-scale, mechanical technology, applied in the field of bioengineering, can solve the problems of difficult control of reversible phase transition conditions, and achieve the effect of easy operation.

Active Publication Date: 2021-02-05
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for fusion proteins with a large number of positively charged ELPs and fusion proteins with mechanical functions, the reversible phase transition conditions are difficult to control, and there is no affinity and ion exchange chromatography for such proteins using large-volume purification columns. method

Method used

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  • A method for large-scale purification of gram-level mechanically functional proteins
  • A method for large-scale purification of gram-level mechanically functional proteins
  • A method for large-scale purification of gram-level mechanically functional proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Sample pretreatment

[0055] Put 2L of Escherichia coli fermentation broth containing SRT protein into a centrifuge, centrifuge in batches, 8000rpm for 20min, collect the precipitate, wash with equilibration buffer A and centrifuge in the same way, repeat three times. A total of 100g of bacterial cells were collected, and then 400ml of equilibrium buffer A was added. After crushing by a high-pressure crusher, centrifuge at 8000rpm for 20min. Dilute to 200mL to finally obtain 200mL of pretreated protein samples.

[0056] 2. Nickel column purification of SRT protein

[0057] Purify the pretreated protein obtained in step 1 through a nickel affinity chromatography column, the column used is HisTrap6XFF 5ml, and the specific purification steps are:

[0058] 2.1 Buffer preparation

[0059] (1) Equilibration buffer A:

[0060]

[0061] Add HCl to adjust pH=8.0

[0062] (2) Elution buffer A:

[0063] Equilibrium buffer A was used to prepare a buffer with a final co...

Embodiment 2

[0119] 1. Sample pretreatment

[0120] Put 20L E. coli fermentation broth containing recombinant Resilin protein into a continuous flow centrifuge, collect the precipitate continuously, wash with equilibration buffer A and centrifuge in the same way, repeat three times. A total of 1 kg of bacteria was collected, and then 5L of equilibrium buffer A was added, crushed by a high-pressure crusher, centrifuged at 8000rpm for 20min, and the obtained supernatant was filtered with a 0.45μm filter membrane to remove bacteria while removing macromolecules, and finally obtained 5L of pre- Process protein samples.

[0121] 2. Purification of Resilin protein by large-volume nickel column

[0122] Purify the pretreated protein obtained in step 1 through a large-volume nickel affinity chromatography column, and the filler used is NiSepharose TM 6Fast Flow high-flow nickel affinity chromatography packing material, the total nickel ion binding capacity of the packing material is not less tha...

Embodiment 3、4

[0182] Similar to the operation method in Example 2, the difference is that the protein is replaced by spidroin and elastin. see results Figure 4-5 .

[0183] The results show, Figure 4 The spidroin protein purity in it reaches 90%, which meets the requirements. Figure 5 The purity of the individual ELPs in is very high, and its purity reaches 99% after analysis, which shows that the expression effect and purification effect of this tag are excellent, and it is extremely effective as a fusion expression tag.

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Abstract

The application belongs to the technical field of bioengineering, and discloses a convenient, rapid method for purifying mechanically functional proteins in large quantities. In the method, the fermentation broth expressing the ELP fusion mechanofunctional protein is pretreated and then purified through a nickel affinity chromatography column, a desalting column, and an SP ion exchange chromatography column. The present invention determines a set of fine purification methods for mechanically functional proteins. After pretreatment, nickel column, desalting column, and ion exchange column, step-by-step treatment is carried out, and pilot scale is carried out to finally obtain mechanically functional proteins with a purity greater than 95%. , and the amount obtained each time reached the gram level. The present invention can be based on a fully automatic protein purification system, which is easy to operate. At the same time, it can realize automatic sample loading, automatic washing, real-time monitoring, online detection, etc. of protein samples, and can finely and specifically adjust the purification process according to the sample status and subsequent purification requirements.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to the invention of a method for mass-purifying gram-level mechanical function proteins. Background technique [0002] Mechanical functional proteins refer to a class of functional proteins with certain mechanical strength, including fibroin, spidroin, arthropod elastin, squid ring tooth protein, etc., mainly fibrous proteins, with a fibrous or thin rod shape, and most of them have a large amount of The β-sheet structure is also the key to its good mechanical strength. However, too many β-sheets will lead to a decrease in solubility, which in turn affects its expression in prokaryotes. [0003] Elastin-like polypeptides (ELPs) are artificial genetically engineered protein polymers that have elastic functions and are very sensitive to ambient temperature. Its structure is mainly composed of pentapeptide repeating sequence units, namely (Val-Pro-Gly-Xaa-Gly, VPGXG...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/22C07K1/18C07K1/16C07K19/00
CPCC07K1/16C07K1/18C07K1/22C07K14/43504C07K14/43518C07K14/43563C07K2319/21
Inventor 刘凯李博李敬敬张洪杰
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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