Under the control of strong transcription and translation signals, the target gene was cloned into T7 phage in pET vector, and its expression was induced by providing T7 RNA polymerase in host cells. Novagen's pet system continues to expand, providing new technologies and choices for expression. At present, * * * includes 36 vector types, 15 different host bacteria and many other related products, aiming at effectively detecting and purifying the target protein.
superiority
It is the most frequently cited system for expressing protonucleoprotein.
In any Escherichia coli expression system, the basic expression level is the lowest.
True "rheostat" control to adjust the expression level.
Provide a variety of different fusion tag and expression system configurations.
Special carrier and host bacteria for soluble protein production, disulfide bond formation, protein transport and polypeptide production.
Many vectors are provided in LIC vector kit for rapid directional cloning of PCR products.
Many host strains are provided in the form of competent cells and can be used for transformation immediately.
Forward pFORCE TM cloning system has the characteristics of efficient cloning of PCR products, forward selection of recombinant and efficient expression of target protein.
Overview of pET system
PET system is the most effective system for cloning and expressing recombinant protein in Escherichia coli. According to the T7 promoter driving system originally developed by Studier et al., Novagen's pET system has been used to express thousands of different protein.
Control the basic expression level
The PET system provides six combinations of vector and host bacteria, and the expression of the target gene can be optimized by adjusting the basic expression level. There is no single strategy or condition suitable for all target proteins, so it is necessary to optimize the selection.
Host strain
After plasmid is constructed in non-expression host bacteria, T7 RNA polymerase gene is usually used to transform the host bacteria (λ DE3 lysozyme) to express the target protein. In λ DE3 lysozyme, T7 RNA polymerase gene is controlled by lacUV5 promoter. It has a certain degree of transcription without being induced, so it is suitable for expressing some genes, and its products have no toxic effect on the growth of host cells. However, when the host bacteria carry pLysS and pLyE, the regulations will be stricter. PLys plasmid encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase, thus reducing its ability to transcribe target genes in uninduced cells. The PLysS host strain produces less T7 lysozyme, while Plyss host strain produces more enzymes, so it is the most tightly controlled λ DE3 lysozyme.
There are 1 1 different DE3 lysogenic host bacteria. BL2 1 and its derived strains are widely used, but they lack lon and ompT proteases. Strain B834 is a methionine auxotroph, so 35 S- methionine and selenomethionine can be used to mark the target protein with high specific activity. BLR is a strain derived from recA, which improves the yield of plasmid monomer and helps to stabilize the target plasmid containing repeated sequences. Two thioredoxin reductase (trxB) mutant strains (AD494, BL2 1 trxB) are beneficial to the formation of disulfide bonds in the cytoplasm of Escherichia coli. Origami TM and OrigamiB strains are double mutations of trxB/gor, and these two enzymes are the key enzymes in the main reduction pathway. The main advantage of Origami and OrigamiB host bacteria is that they can form correctly folded protein containing disulfide bonds. The new Rosetta TM strain supplemented the tRNA of four rare codons of Escherichia coli, and improved the low expression of some eukaryotic protein due to different codon usage frequencies. Other strains include K- 12 HMS 174 and NovaBlue, which are recA-like BLR. These strains can stably express some target genes, and their products may lead to the loss of DE3 phage. NovaBlue is a useful close host strain because of the high affinity lacIq repressor protein encoded by F-appender. In addition, Novagen also provided λ DE3 lysogenic kit for preparing new expression host bacteria with other genetic backgrounds. Another alternative method to express highly toxic genes or prepare new λ DE3 lysosomes is to provide T7 RNA polymerase through l CE6 infection. Although this is not as convenient as inducing λ DE3 lysozyme by IPTG, this strategy is also preferred in some applications.
Highly strict T7 lac promoter
In addition to selecting three basic expression stringency at the level of host bacteria, T7 promoter in pET system itself provides two different stringency choices: ordinary T7 promoter and T7 lac promoter. T7 lac promoter contains a 25bp lac operating sequence at 17bp downstream of the promoter region. The binding of lac repressor protein at this site can effectively reduce the transcription of T7 RNA polymerase, which provides a second lacI-based mechanism (except lacUV5) to inhibit the basic expression of λ DE3 lysozyme. PET plasmids containing T7 lac promoter also have their own lacI, which ensures that enough repressor proteins are bound to the manipulation gene site.
In practical application, in order to obtain the highest protein yield, it is usually necessary to test various combinations of vector/host bacteria.
Control induced expression level
In many cases, the expression of protein with the best activity and solubility depends on the background of host cells, culture conditions and appropriate vector configuration. Usually, the conditions with the highest activity of the target protein are inconsistent with the conditions with the highest yield. PET system not only controls the basic expression of T7 RNA polymerase according to the combination of vector and host bacteria, but also provides a real "rheostat" control for the expression of target protein according to the concentration of inducer (IPTG). LacY mutation of tuner and OrigamiB host bacteria makes this control possible.
Select a pet carrier
All pET vectors are from pBR322, but they are different in leader sequence, expression signal, fusion tag, related restriction sites and other characteristics. There are two types of pET plasmids, namely transcription vector and translation vector:
Transcription vectors (including pET-2 1, pET-23 and pET-24) express the target RNA, but do not provide translation signals. They are used to express protein of target gene with bacterial translation signal. (Note: Transcription vectors are distinguished by the missing letter suffix after the name. )
The translation vector contains an effective translation initiation signal designed for protein expression. Many vectors have cloning sites in reading frames A, B and C, which correspond to GGA, GAT and ATC triplet of BamH I site respectively.
Select key points
Choosing a pET vector for expression usually involves many factors. Consider the following three main factors:
The use of protein expressed.
Express the known information of protein.
Cloning strategy
Protein expressed by pET vector has many uses. For example, protein with analytical expression level can be used for activity research, mutant screening and characterization, ligand interaction screening and antigen preparation. A large number of active proteins are used for structural research, reagent or affinity matrix preparation. Many vectors are suitable for expressing analytical proteins for screening or antigen preparation, but only when the combination of vectors, host bacteria and culture conditions is very suitable can they be used for mass purification. If continuous high yield of active protein is needed, various combinations of vectors, host bacteria and culture conditions should be tested to find the best results.
Any known information about the target protein is helpful to the selection of vectors. For example, the activity of some protein requires that there is no exogenous sequence at one or both ends. Many pET vectors can clone non-fusion sequences. However, if a specific translation initiation sequence cannot be effectively used in E.coli, the expression level may be affected. In these cases, fusion proteins can usually be constructed with efficiently expressed amino-terminal sequences, and then the fusion sequences are digested with site-specific proteases after purification. LIC (ligation-independent cloning) strategy is particularly useful for this method, because cloning operation can remove all amino-terminal carrier coding sequences through enterokinase and factor Xa.
Because of the compatibility of restriction sites and reading frames, cloning strategy will also affect the choice of vectors. Because many pET vectors have the same restriction site configuration, it is usually possible to clone the target gene prepared at one time into several vectors. There are different considerations when adopting PCR cloning strategy. Therefore, it is recommended to use the LIC vector kit. Inserts can be prepared by PCR without restriction of digestion vectors or inserts.
Solubility and cell localization
Considering the application and cloning strategy of the target protein, it is very important to determine the cell localization and solubility of the target protein. In many practical applications, it is often necessary to express soluble active proteins.
The solubility of specific target protein depends on many factors, including their respective protein sequences. In many cases, solubility is not a phenomenon of existence or non-existence, and vectors, host bacteria and culture conditions can be used to increase or decrease the proportion of soluble or insoluble forms obtained. PET-32 vector series fused the target sequence with thioredoxin (Trx). Tag), which can usually increase the proportion of soluble protein. The newly launched pET-43. 1 series has a fusion partner-NUS. Tag, an Escherichia coli protein with high solubility when overexpressed, was obtained through a large number of systematic screening, thus further improving the solubility of the target protein. In addition, trxB mutants AD494 and BL2 1 trxB, or trxB/gor origamit and OrigamiB strains can be used to form disulfide bonds required for the correct folding and activity of many eukaryotic protein in cytoplasm. Low temperature induction (15-25℃) can also increase the proportion of soluble target protein.
Another strategy to obtain soluble active protein is to secrete the protein into the cell periphery, which provides a more suitable environment for folding and disulfide bond formation. In order to achieve this goal, carriers with signal peptides are usually used.
Some purification strategies can optimize the yield of insoluble inclusions in cytoplasm. Inclusion bodies are extracted and dissolved, and then the target protein is refolded in vitro (for example, using Novagen's protein folding kit). This process usually produces a high yield of initial protein and prevents protein degradation in the host cell. However, the efficiency of refolding into active protein varies greatly with different protein, and may be quite low. PET-3 1b (+) vector is specially designed for producing insoluble fusion proteins, which provides an effective method for producing small proteins and peptides.
Fusion label meeting different needs
If the fusion sequence does not affect the application, S.Tag and T7 are used to produce the fusion protein. Label a, his. Tag a and HSV. Tag a will facilitate the subsequent operation and easily pass the protein hybridization detection. These peptides (the fusion sequence is very small) and their detection reagents are extremely specific and sensitive. Goods and services tax. Tag, S tag and T7. The tag sequence can be used for affinity purification by using the corresponding resin and buffer kit.
By using S.Tag and GST, crude or purified fusion protein can be accurately quantified. Tag analysis kit. The FRETWorks S.Tag detection kit with a new substrate can detect the fusion protein less than 1fmol by fluorescence.
His. Tag a sequence is very useful as a fusion partner of purified protein, especially for those proteins expressed in inclusion bodies, which can make affinity purification possible under the condition that the dissolved protein is completely denatured.
CBD。 Tag A is very useful in low-cost affinity purification. They are also particularly suitable for refolding (especially pET-34b (+) and 35b (+) and CBD clos. Tag a sequence). Because only the correctly refolded CBD binds to the cellulose matrix, the CBIND affinity purification step can remove the incorrectly folded molecules from the preparation. Any label can be used to immobilize the target protein, but it is more suitable for this purpose because of its non-specific binding to cellulose matrix and low biocompatibility.
National university. Label, Trx. Label and GST. The tag sequence is used to increase the solubility of its fusion partner. National university. Labels and transactions. The label carrier is compatible with origami host bacteria, which is beneficial to the formation of disulfide bonds in cytoplasm.
See various fusion labels and the corresponding list of pet carriers. Some pet carriers carry several fusion tags as 5' fusion partners. In addition, many vectors express fusion proteins with different polypeptide tags at the end through the reading frame of the target gene sequence. Using a vector containing protease cleavage sites (thrombin, factor Xa and enterokinase) between the 5' tag and the target sequence, one or more tags can be selectively removed after purification. PET-30 Ek/LIC, pET-32 Ek/LIC, pET-34 Ek/LIC and pET-36 Ek/LIC are good representatives of cell localization and affinity tag configuration. PET Ek/LIC vector combination kit includes all four ready-to-use vectors and can be directly used to construct several target gene configurations.
PET NusA fusion system 43. 1
Production of soluble active protein in Escherichia coli
The newly introduced pET NusA fusion system is designed to clone and express the polypeptide sequence fused with 495aaanusa (NUS) at high level. Label) protein. Based on the solubility modeling of more than 4000 kinds of protein in the database, NusA protein was proved to have the highest solubility. In the experiments with four different NusA fusion proteins, more than 85% of the expressed proteins were soluble. PET-43. 1 vector contains Nus. Tag fusion partner is compatible with trx/gor mutants Origami and OrigamiB, which is beneficial to the formation of disulfide bonds in cytoplasm. Using the combination of pET-43. 1 vector and these trx/gor host bacteria, it is possible to obtain disulfide-bonded protein.
superiority
Highly soluble n-terminal Nus. Tag fusion partner
His upstream. Tag a, S. tag and optional C-terminal HSV. Label a, his. Tag fusion tag.
Thrombin and enterokinase cleavage sites
Polyclonal sites are located in all reading frames.
Compatible with AD494 and Origami host strains, which can promote the correct folding of the expressed protein.
Competent cell of pet host strain
All the host bacteria of pET system are provided in the form of pre-detected competent cells, which can be used for transformation immediately.
(DE3) indicates that the host is λ DE3 lysozyme with a copy of T7 RNA polymerase gene controlled by lacUV5 promoter on its chromosome. This strain is suitable for producing protein from the target gene cloned into pET vector. Host bacteria named pLysS and pLysE carry pET-compatible plasmids encoding T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase. Cells with pLysS produce a small amount of lysozyme, while pLysE host bacteria produce a large amount of enzymes. These strains are used to inhibit the basic expression of T7 RNA polymerase before induction, which can stabilize the pET recombinant encoding the target protein affecting cell growth and vitality. The host bacteria with pLacI produced additional lac repressor protein, which inhibited the basic expression of pETBlue and pTriEx vectors. λ DE3 lysogenic kit is used to prepare new expression host bacteria with other genetic backgrounds.
AD494 strain is a mutant strain of thioredoxin reductase (trxB), which can form disulfide bonds in cytoplasm, providing potential for producing correctly folded active proteins. TrxB mutation can be selected by kanamycin, so this strain is recommended for plasmid with ampicillin resistance marker bla.
B834 is the parent strain of BL2 1. These protease-deficient host bacteria are methionine-deficient, and the target protein can be labeled with 35 S- methionine and selenomethionine with high specific activity, which can be used for crystallographic research.
BL2 1, the most widely used source of host bacteria, has the advantage of lacking lon and ompT protease.
The strain BL2 1 TrxB has the same thioredoxin reductase mutation (TrxB) as the strain AD494 in the absence of protease BL2 1. TrxB hosts are beneficial to the formation of disulfide bonds in cytoplasm, so using them can increase the correctly folded protein component. TrxB mutation can be selected by kanamycin, so this strain is recommended for plasmid with ampicillin resistance marker bla.
RecA-derived strains with BLR of BL2 1 can improve the yield of plasmid monomers and help to stabilize target plasmids containing repetitive sequences or their products, which will lead to the loss of DE3 phage.
HMS 174 strain provided recA mutation on the background of K- 12. Like BLR, these strains can stabilize some target genes, and their products can lead to the loss of DE3 phage.
NovaBlue is suitable for K- 12 strain as the initial cloning host strain, which has high transformation efficiency, blue/white spot screening ability (with suitable plasmid) and recA endA mutation leading to high yield and high quality plasmid DNA. DE3 lysozyme of NovaBlue is a very useful close host strain because of the existence of lacI q repressor protein encoded by F-attach.
Origami is a host strain derived from K- 12. Thioredoxin reductase mutation (trxB) and glutathione reductase (gor) genes are mutations, which can greatly enhance the formation of disulfide bonds in cytoplasm. Studies have shown that even if the overall expression level is similar, the active protein expressed by Origami (DE3) is 10 times higher than that of other host bacteria. Origami host strain is compatible with ampicillin resistant plasmid and can be used as pET-32 vector. Thioredoxin labeling can further enhance the formation of disulfide bonds in cytoplasm. TrxB and gor mutations can be selected by kanamycin and tetracycline, respectively, so this strain is recommended for pET plasmid with ampicillin resistance marker bla.
Origami B host strain is derived from BL2 1 lacZY mutant and has the same TrxB/gor mutation as the original Origami strain. Origami B strain combines the advantages of BL2 1, Tuner and Origami host strains. TrxB and gor mutations can be selected by kanamycin and tetracycline, respectively, so this strain is recommended for pET plasmid with ampicillin resistance marker bla.
Rosetta host strain is derived from BL2 1, which can enhance the expression of eukaryotic protein of rare codons in Escherichia coli. The strain supplemented the tRNAs of codons AUA, AGG, AGA, CUA, CCC and GGA with compatible chloramphenicol resistance plasmids. In this way, Rosetta strain provides "universal" translation, thus avoiding the expression restriction caused by the frequency of codon usage in Escherichia coli. TRNA gene is driven by its natural promoter. In Rosetta (DE3) pLysS and Rosetta (DE3) pLacI, the rare tRNA gene exists on the same plasmid as T7 lysozyme and lac repressor gene, respectively.
Tuner strain is a lacZY deletion mutant of BL2 1, which can regulate the protein expression level of all cells in the culture. Lac permease (lacY) mutation makes IPTG enter all cells of the population evenly, so it is concentration-dependent and induces expression evenly. By adjusting the IPTG concentration, the expression can be adjusted from a very low level to a very strong and completely induced expression level (usually related to pET vectors). Low-level expression can sometimes enhance the solubility and activity of protein, which is difficult to express. Tuner (DE3) pLacI strain is compatible with the expression of pETBlue and pTriEx vectors.
Purification of recombinant protein
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