Low RNA integrity, is the result necessarily poor? It's time to uncover the truth. My younger brother commented on Professor Natural Plants, and I also extended five kinds of mouse models in statistical tumor immunity research at the doctor's point, so that you could check the literature for half a month less. The difficulties and solutions of mammalian cell expression have been sorted out for you. When you do protein purification, failure is inevitable. Either the protein cannot be expressed, the expressed protein cannot be detected, and the protein cannot be hung on the column ... In order to better help everyone in the field of protein purification,

Portal: The difficulties and solutions of mammalian cell expression have been sorted out for you.

Among them, Strep-tag tags (including Strep-tag? Type II or Double Streptococcus Label? ) with streptococcus label? Purification system has attracted more and more researchers' attention because of its mild purification process, high purity of target protein and compatibility with various expression systems.

Advantage principle

Third generation streptococcus label? Protein purification system, through double-stranded tags? Label with streptococcus. XT, compared with the previous generation (Strep-tag? Two: streptomycin? ), the affinity is close to * * *, but the bonding is still reversible, so the purity is high (purity >; 95%), the yield of the target protein is high, and the activity of the target protein can be maintained. Moreover, the system still has good purification performance in the environment with surfactant, and washing the column for many times will not cause the loss of the target protein, even if it is a small amount of protein or membrane protein, it has good purification efficiency.

Figure 1

Application case

In the research example, the author used Strep-Tag? Latex scavenging protein (LCPs) 1 was successfully expressed and purified. Lcp is a kind of rubber oxygenase, and it is one of only three extracellular rubber oxygenases that have been studied and characterized by biochemistry. It can be used by Gram-positive Streptomyces K30(LcpK30) to grow on natural rubber. Because natural rubber is widely used as daily necessities such as sealed bags, gloves and tires, the generated waste also enters the environment permanently. The main component of cis-1, 4 polyisoprene can be cracked by oxygenase. After the reaction, the low-cluster isoprene with aldehyde group and ketone group can be used as the synthesis precursor of biofuel or fine chemicals. Therefore, it is of great significance to study rubber oxygenase.

Let's compare streptococcus markers. II- LcpK30 matches Strep-Tactin respectively? High capacity and strep-tact? Purification effect of XT.

Figure 2

SDS-PAGE analysis showed that Strep-Tactin was used. High capacity and strep-tact? XT, a large number of high-purity protein can be obtained; Relatively speaking, with streptomycin? High capacity will lose a small amount of protein in the last elution step. Use streptomycin? XT can avoid the loss of protein. Therefore, the protein yields of the two resins are different (Figure 3). Compared with high-capacity streptopeptide, streptopeptide? XT can purify about 13% Strep-Tag? II-LcpK30, with higher efficiency and lower price.

Figure 3

Use the second generation of streptomycin? Compared with the third generation of streptomycin? XT and Strep-Tag? II has high affinity, which can make the washing step more intense without losing the target protein. Streptococcus tactical protein. XT can obtain extremely high protein purity under physiological conditions, and its high affinity can quickly purify Strep-Tag? The mild elution conditions of II-LcpK30 can keep the activity of the enzyme, which provides a guarantee for successfully determining its three-dimensional crystal structure.

Not only that, streptomycin? XT can also be used for purification under denaturing conditions, or WB/ELISA to detect target proteins, immobilize target proteins, detect protein interactions, or further screen therapeutic proteins and industrial enzymes. , has a wide range of applications.

Reference (scroll down)

1.r？ Ther W, Birke J, Grond S, et al. Production of functionalized low-cluster isoprene by enzymatic cracking of rubber. Microbial biotechnology 43:1238–1433 [j]. 20 17.

2. Burke j, r? Ther W, Jendrossek D. RoxB is a new rubber oxygenase [J] which combines the characteristics of rubber oxygenase RoxA and latex scavenging protein (Lcp). Applied and Environmental Microbiology, 20 17: AEM. 0072 1- 17.

3. Burke j, r? Functional identification of rubber oxygenase in soil and marine myxobacteria [J]. Applied and Environmental Microbiology, 20 13: AEM. 02 194- 13.

4. Burke j, r? Latex scavenging protein of Streptomyces. K30 is a type B cytochrome, and its biophysical properties are different from those of rubber oxygenase A(RoxA)[J]. Applied and Environmental Microbiology, 20 15: AEM. 00275- 15.

Article source: International Bar Association

Image source: iba

Title source: Station Cool Luohai

Subject: Streptococcus labeling, protein purification.