Bacterial expression systems for exogenous protein production
Bacterial expression systems for exogenous protein production
Bacterial expression systems are available in a wide variety of vectors and host bacteria, and the short proliferation time of most engineered bacteria not only facilitates rapid evaluation of experimental results, but also reduces the stringency of technical and equipment sterility requirements. Many of these advantages inherent in laboratory-scale processes are also inherent in large-scale automated production processes, with a few simple adjustments.
Operation method
Bacterial expression systems for exogenous protein production Move I. Production of exogenous proteins using E. coli bacteria There are a growing number of bacterial expression systems available for the production of exogenous proteins. Factors affecting the choice of a particular expression system include the natural nature of the target protein, the experience of the user and the intended use of the product. E. coli is the most commonly used bacterial expression system for the expression of exogenous proteins. After more than a century of extensive research on E. coli, a great deal of information has been gained by researchers in terms of regulatory mechanisms and the function of host auxiliary proteins that may affect the outcome of expression. In addition, there is a wide range of sources for methods and techniques required for laboratory and commercial protein production. For many protein preparation projects, these available resources and very low technical requirements make E. coli the host of choice for initial protein expression and screening. Therefore, we will use E. coli as a model to introduce specific methods and techniques for protein production. After presenting specific techniques for protein expression using E. coli, we will also explore other available bacterial expression systems. Many of the core concepts and techniques for protein production using E. coli presented here can be directly applied to other bacterial expression systems. The successful use of a bacterial expression system relies on an organized series of steps listed in chronological order in Table 12.1. The initial stage of protein production is not the initiation of experiments, but rather the clarification of the complete set of requirements for the protocol and the analysis of the sequence identity and biochemical properties of the expression target. These steps are critical to the ultimate success of the protocol and contribute to a realistic assessment of the cost and likelihood of success of the project. In order to maintain continuity with the experimental section, we present these steps briefly, as they are described in more detail in the sections of this chapter. The intended use of the expression product is important in the selection and prioritization of expression systems. For some applications (e.g., functional profiling or drug screening), preserving the natural function and properties of the protein is necessary; however, this is less relevant if it is to be used as an immunogen. Other practical considerations include protein yield, time constraints, or production costs. Defining the needs of the project is essential to ensure that the appropriate expression system, cloning strategy, and production strategy for the desired product are selected (Dieckmanetal., 2006). The biochemical and biological properties of target proteins are essential for the selection of an expression strategy, and they can be used to make initial predictions of expression results and product solubility. Analysis of the primary sequence allows the integration of a set of properties to predict secondary structure, biological localization (membrane, cytoplasm, periplasmic lumen, or extracellular), family classification (folding and structural domains), and inferred biochemical properties (isoelectric point, disordered region, ligand). Some features of the target protein, such as the high likelihood of the presence of a transmembrane helix, may necessitate the use of a system designed for the expression of membrane proteins or require the use of a strategy that clones individual structural domains in order to express the soluble portion of the protein. Using the characteristics of the target protein to guide the selection of expression hosts and the construction of vectors can help to obtain mature, appropriately localized proteins. Sequence information can be supplemented using experimental and historical data on the target protein or homologous proteins, thus contributing to further optimization of the expression system. For example, if it is known that a protein requires a certain cofactor to maintain proper functionality, then a specific host must be selected to ensure that a fully functional expression product is obtained. A case in point is the expression of cytochrome c, which benefited from the use of genetically engineered E. coli expressing a cofactor protein that covalently attaches heme to the polypeptide chain of the cytochrome precursor (Londeretal., 2008). In practice, it is very difficult to predict the outcome of a protein expression experiment in the absence of historical expression data for the target protein or similar proteins. It is common practice to use several expression systems, starting with the simplest and most cost-effective one and failing with a more complex one. There are several options for cloning target sequences, which can usually be categorized into tandem and parallel systems. Tandem systems are widely used and provide a variety of methods for generating target sequence/vector matching ends for the construction of expression vectors, such as those utilizing restriction endonucleases to generate target sequence/vector matching ends. The disadvantage of this method is that the cutting strategy of restriction endonucleases needs to be validated for each target sequence and vector. Researchers are increasingly interested in parallel systems, or universal cloning techniques, which allow easy transfer of target fragments into a variety of vectors and expression systems without regard to the sequence of the target fragment (Table 12.2). Examples of such systems include Gateway (EspositoetaL, 20 0 9), Infusion (Zhuetal., 2007) and ligationindependent cloning (LIC) (AslanidisanddeJong, 1990; Haunetal., 2007). Haunetal., 1992). The advantage of this method is the ability to clone a single target fragment into several different vectors, allowing for the simultaneous evaluation of multiple expression strategies in a cost-effective manner. LIC is a cost-effective and particularly suitable technique for bacterial expression systems because the reagents used for cloning are not patented and are available from several suppliers. In the LIC technique, specific nucleotide sequences are added to the PCR primers, which allows the cloning of any gene regardless of the DNA sequence. Vectors and PCR fragments with matching ends can be obtained by treatment with T4DNA polymerase with the participation of a special nucleoside triphosphate. This treatment produces 10-15bp sequence-complementary single-stranded protrusions on the vector and PCR fragments, which are sufficiently strong after annealing to be used for transformation without ligation. This method allows the PCR primers to be designed in a consistent and directional manner, resulting in high cloning efficiency. Although commercial vectors are available, the method has been used in several large-scale cloning projects that can provide vector resources to individual researchers. The procedure described below was developed to use a set of vectors designed by the Midwest Center for Structural Genomics (Eschenfeldtetal., 2009). This technique is generally applicable to other LIC-compatible systems as well, as long as the specific sequences added to the amplification primers are adjusted. 1. Add appropriate LIC-specific nucleotide sequences to the target sequence-specific PCR primers (e.g. forward primer: TACTTCCAATCCAATGCC; reverse primer with stop codon: TTATCCACTTCCAATGTTA). 2. The method can be amplified and performed using a microtiter plate (96 reactions), but can be adjusted to any number of reactions. Prepare a LIC reaction mixture sufficient for % well plates by mixing the following reagents. ① 465uL 10 X T4 DNA polymerase buffer. Most distributors offer T4 DNA Polymerase Buffer as a substitute for the LIC Reaction Buffer described in this section. In comparison, we found that different common T4 DNA polymerase buffers affect the cloning efficiency of the final product by less than 25%. ② 465 uL of molecular biology grade 25 mmol/L dCTP (Promegacat. no. U1221). ③ 228uL 100 mmol/L DTT (Clithiothreitol) solution (Novagencat.no.70099). ④ 60ul of water. ⑤ 250 units of T4 DNA polymerase (LIC quality, approx. 2.5 U/uL, EMD Biosciences/Novagen). 3. Place the mixture on ice and add the T4 DNA polymerase before use. Pipette several times to evenly distribute the enzyme in the reaction mixture. 4. Add 10.4uL of LIC reaction mixture to each well of a polypropylene 96-well plate. 5. Add 30yLUO?100ng) of purified PCR fragments to the LIC reaction mixture and mix by pipetting several times. Incubate at room temperature for 30 min. Studies using multiple fragment-to-vector ratios have shown that the annealing reaction is very tolerant of variations in the amount of target DNA fragments. 6. Incubate for 20 min at 75°C on a heating block to inactivate T4DNA polymerase. 7. On another 96-well plate, anneal 1~2 ul of T4 polymerase-treated PCRLIC fragments and 4 dozens (20~50ng) of T4 polymerase-treated LIC vector. 8. Incubate the annealing reaction at room temperature for 5?10 min. 9. Transform approximately 50 E. coli receptor cells using the full annealing system, and select the transformed clones (SambrookandRussell,2001). After heating, the LIC plates were stored in the refrigerator at 4°C until needed.The preparation of LIC compatible vectors is similar to the above procedure, but with the use of base complementary dGTP (Eschenfeldtetal., 2009). At this stage, the constructed vectors can be characterized by sequence analysis or after completion of expression and solubility analysis. Most E. coli expression vectors are designed for intracellular expression. These vectors are engineered with different selectablemarkers, bacterial promoters, plasmidreplicationorigin, localizationsignal and fusion tags (Table 12.2). The method we describe here applies to the T7 promoter (Novagen, pET vector family), which is capable of overexpressing target proteins at levels comparable to those of the most abundant natural proteins in Escherichia coli (Studier and Moffatt, 1986). E. coli strains capable of expressing T7RNA polymerase [e.g. BL2KDE3)] are required for protein expression using the pET family of vectors. Variations of these strains are capable of co-expressing tRNAs with rare codons (Carstens, 2003), co-co-factors essential for protein folding (BaneyxandPalumbo, 2003), or proteins that contribute to the formation of disulfide bonds in the cytoplasm and promote active folding of recombinant proteins (PrinzetaL, 1997). The scheme here summarizes the process of expressing target proteins using IPTG and inducible systems. 1. Inoculate the monoclones in 2 mL of medium containing the appropriate antibiotics. The volume of the tube used should be at least 5 times the volume of the medium to ensure adequate aeration. 2. Incubate the samples at 37℃ at 250 r/min until the OD600nm reaches 0.4~0.8. The culture solution becomes cloudy but not completely turbid. Usually it takes 3~4 h for BL21(DE3) to reach this state. 3. Add 20ul of 100 mmol/L IPTG to each culture (1 mmol/L final concentration). Place the induced cultures back into the bacterial incubator at 37°C, 250r/min for 4 h. After 4 h of induction, the cultures will become completely cloudy. 4. Cultures should show clear protein expression after 3?8 h of induction at 37°C. Remove the sample and analyze it as described below. Confirmation of the expression of a target protein typically involves an assessment of protein expression and protein solubility, as well as qualitative confirmation of the expected protein size. Most E. coli systems used for exogenous protein expression can produce sufficient levels of the target protein to allow analysis of protein expression/solubility by denaturing gel electrophoresis (Figure 12.1). This method is inexpensive, relatively simple, and provides quick results. Proteins with very low levels of expression or solubility may require more sensitive detection methods such as immunoblotting. 1. Remove 200uL of inducer from each sample into a clean microcentrifuge tube. centrifuge the organism at 14,000 IVmin. The organism will form a dense precipitate with a clear supernatant. The remaining culture can be used for soluble mass analysis of the target proteins as described in Section 10.1 of this chapter. 2. Pour off or aspirate the unwanted medium, carefully retaining the bacterial precipitate. Add 502XSDS Sampling Dye and resuspend the organisms by blowing several times. 3. Boil the sample for 5 min and allow to cool before sampling. For a 17-well, 8 cm X 10 cm, kosmos-stained gel, 5 uL of sample is usually sufficient. 1. Form a precipitate from the remaining bacterial culture by centrifugation at 3500r/min for 10 min. Carefully pour off the unwanted medium and dip the residual liquid into a paper towel to dry. 2. -80°C freeze the precipitate. Prepare sufficient lysis buffer for all samples [final concentration: 300 mmo/L NaCl, 50 mmol/L Na2Po4, protease inhibitorCocktail (Sigma), 120 kU/mL recombinant lysozyme (rLysoZyme, Novagen) and 25U, mL nuclease (Benzonase, Novagen)]. mL nuclease (Benzonase, Novagen)]. Alternatively, Bugbuster (Novagen) or frPER (Pierce) may be used instead of lysis buffer. Follow the reagent instructions and use the reagents appropriate for 2 mL cultures. 3. Remove frozen samples and thaw slightly. Add I80 Lysis Buffer to each sample, cover tightly and vortex to resuspend the precipitate. 4. Replace the samples at I80°C for 5 min, then remove and incubate at room temperature until the ice is completely dissolved. Repeat the freeze-thaw once more. The sample will either become clear or remain in a cloudy pool. 5. Centrifuge at 3500 r/min for 10?15 min to precipitate the bacterial debris. 6. Remove 50fxL of supernatant from the top of each sample, being careful not to remove any of the bacterial debris. 7. Add 60 fxL2XSDS Sample Dye to the supernatant and boil for 5 min. Analyze the samples by denaturing gel electrophoresis. For a 17-well, 8 cmX10 cm, kosmos-stained gel, 5 porphyrin samples are usually sufficient. 8.(Optional) To detect the insoluble fraction in the bacterial lysate, remove all remaining supernatant from the bacterial lysate fractions from step 7. Be careful not to disturb or remove the precipitate. 9. Add 300juLIXSDS Sampling Dye to the precipitate, cover tightly and vortex until the precipitate is completely resuspended. 10. Boil the sample for 5 min and allow to cool slightly before loading onto the SDS-PAGE gel. For a 17-well, 8 cmX10 cm, kosmos-stained gel, 3 samples are usually sufficient to visualize expression. Using the same culture system when assessing expression and solubility can compensate for differences in expression levels. Protein expression in E. coli can typically exceed 80%, but protein solubility is the main limiting factor in this system, which is related to the target protein itself (Figure 12.1). After SDS~PAGE analysis, if no stained bands of the target protein are detected at the correct molecular mass position, then it can be considered "not expressed" or "insoluble". On the contrary, it is considered to be "expressed" or "soluble". This is a qualitative assessment and must be followed by sequence analysis and/or mass spectrometry to confirm the identity of the ^peptide. We used a relative density sequencing method, whereby the staining density of the target protein is compared to the usual staining density of natural proteins in E. coli. Target bands that are visible but less densely stained than most E. coli protein bands are labeled as level 1 or low expression/low solubility. levels 2 and 3 (medium or high expression/high solubility) indicate staining intensities comparable to or higher than those of proteins highly expressed in E. coli, respectively. One drawback of IPTG induction is the need to monitor bacterial growth for optimal induction conditions. This disadvantage becomes particularly acute when detecting the expression of a large number of clones. In this case, differences in growth rates can be observed, making it difficult to obtain optimal induction conditions for all expressed clones. Self-inducible systems provide us with a way to overcome these difficulties and simplify the expression protocol (Studier, 2005). Several self-inducible systems have been reported that can utilize pET and other IPTG-induced bacterial expression systems to provide high levels of protein expression (Blotnmeletal., 2007). 8%?12% of the bacterial proteome is not localized in the cytoplasm. Exogenous expression strategies for these proteins include amplification of the non-signal peptide portion of the coding region, using cytoplasmic expression vectors or periplasmic expression vectors, and proceeding through the standard E. coli cloning and expression process, resulting in expression similar to that of cytoplasmic proteins. This method has also been successfully applied to certain eukaryotic proteins containing disulfide bonds. Proteins can be localized in the periplasmic space of E. coli by adding a suitable localization signal (e.g., the E. coli PelB signal sequence) to the N-terminus of the target protein. Since the periplasm accounts for only 20%?40% of the total volume of the organism, in general, periplasmic expression is generally less abundant than cytoplasmic expression. This conclusion can be reached by directly comparing the expression levels of target proteins cloned into cytoplasmic expression vectors (Fig. 12.2, target proteins 4?6) with those cloned into periplasmic expression vectors (Fig. 12.2, target proteins 10?12). 1. Inoculate the monoclonal in 2 mL of medium containing suitable antibiotics. The holding volume of the tube used should be at least 5 times the volume of the culture medium to ensure adequate aeration. 2. Incubate at 30°C at 250 r/min until the OD68jnm reaches 0.4?0.8. The medium should become cloudy but not completely turbid. Usually BL2KDE3) organisms need to grow for 3?5 h to reach this state. 3. Lower the temperature of the culture vessel to 19°C and incubate for at least 30 min to equilibrate the organisms. The OD value of the culture will continue to increase during the temperature change. 4. Add 20 dozens of 100 mmol/LIPTG to each culture (final concentration is Immol/L). Return the culture system to the culture vessel and incubate overnight at 19°C, 250r/min. After induction overnight, the culture solution will become completely turbid. 5. Cultures will show a small amount of expression after 4 h of induction at 19°C, and obvious expression after 12~16 h. The culture should be induced at 19°C and 250r/min overnight. If it is necessary to confirm that a target protein is localized in the periplasm, then the osmotic shock method can be used to analyze the periplasmic fraction separate from the cytoplasmic fraction. A number of techniques are available for releasing proteins from the bacterial periplasm. Addition of chloroform (Amesetal., 1984) or polymyxin B (Dixon and Chopra, 1986) can release proteins from the periplasm. The method described below is inexpensive and uses commonly available laboratory reagents (NeuandHeppeU1965;NossalandHeppeU1966). Addition of lysozyme to the osmotic shock SETC (sucrose, EDTA, Tris-HCl) buffer removes more of the outer wall and components of the bacterium (BirdsellandCota-Robles1967;MalamyandHorecker1964); the protoplasts produced by this step are very easy to lyse (NeuandHeppeU1965;NossalandHeppeU1966). spheroplast) produced in this step are very susceptible to lysis and therefore cytoplasmic proteins may contaminate periplasmic fractions. For lysogenic organisms, the addition of 0.5 mmol/L MgCl2 to the cold water shock solution stabilizes the protoplasts (NeuandChou, 1967). The method we present works for most proteins that rake the periplasm, but may need to be optimized for some proteins that target the periplasm. Comparison of the amounts of target proteins in SET and in water in Fig. 12.3 illustrates that the extraction results are different for different proteins. However, soluble exogenous proteins are enriched in the shock fraction compared to background host proteins (Fig. 12.3). This method can be scaled up as a first purification step to reduce background proteins and bacteriophage debris. There are many different bacterial expression systems that can be used to express exogenous proteins. In many cases, the choice of bacterial expression system is usually determined by the properties of the target protein. Although E. coli can be used for the exogenous expression of polyhemecytochromes (nmltihemecytochromes), another host is often chosen for the expression of complexcytochromes containing multiple heme moieties. S/iewaneWaonei<ie7m\s) is a Gram-negative bacterium commonly used for the production of this particular protein. The genome of this organism encodes a large number of predicted cytochrome c genes and contains auxiliary proteins that facilitate the proper processing of cytochrome c into a mature protein (TakayamaandAkutsu, 2007). An engineered Pseudomonas aeruginosa (_Psewc?omas/ZM 〇? -e?:e?w) strain (Pfenex, DOWChemicalCa) has been developed as an exogenous protein expression system for high-volume fermentation and high-throughput small-scale screening of target proteins. This modified Gram-negative bacterium has multiple metabolic functions and a microbial secretion system that allows the expression product to be located in the cytoplasmic, periplasmic, or extracellular space. Expression of proteins using non-pathogenic Gram-positive bacilli provides us with an alternative to Gram-negative host bacteria (Schmidt, 2004). These organisms contain a naturally efficient secretion system that allows for the expression of proteins directly in the culture supernatant, which allows for higher yields while facilitating purification. The most commonly used strain is the engineered Bacillus subtilis (BadWws), which lacks the genes for extracellular lipolytic enzymes and proteases, thus improving the stability of exogenous proteins. BaciZZwsOTegaferiwm has several large plasmids and is known for its ability to stably replicate and maintain these plasmids. Its expression strains typically have low intrinsic protease activity, and a number of features that combine to achieve stable, high yields of proteins. Membrane proteins make up a sizable portion of the proteins encoded by the genome, but remain a challenge for protein expression. In addition to E. coli (NeophytouetaL,2007;ShawandMiroux,2003), researchers have developed several other membrane protein expression systems to express intact membrane proteins. The Gram-positive lactic acid bacteria, Lactococcus lactis (LartococcwsZartt's), grow at high densities and are suitable for large-scale membrane protein overproduction (Kunjietal., 2003). Several nutritionally deficient strains and inducible expression systems regulated by polycyclicpeptidenisin are available. Functional screens for the identification of membrane proteins can be performed using whole bacteria because the ligands can act directly on membrane proteins expressed at the cell membrane. The photosynthetic bacterium Rhodobacter spp. has been engineered for the exogenous expression of full-length membrane proteins (Laibleetal., 2004). This system is unique in that it can integrate exogenous membrane proteins into its own system of endogenous membrane proteins by synthesizing new membranes at the same time as expressing the exogenous membrane proteins. The formation of these new cellular membranes causes an inward concavity of the cytoplasmic membrane, allowing the integration of exogenous membrane proteins. A very strong point of the membrane protein expression system of the genus Erythrobacter is that the proteins expressed in all cases are localized to the membrane in almost equal amounts. Expression and purification of membrane proteins can be difficult, and the function of proteins produced with exogenous expression systems must be carefully verified. In addition to the use of different bacterial hosts, optimization of exogenous expression systems can be achieved by controlling the vector and induction conditions. One such method that has proven successful for soluble expression of proteins uses the cold shock expression system with the pCold vector (Qingetal., 2004). This system utilizes the cold shock response of E. coli cells in order to inhibit the production of endogenous proteins while at the same time enhancing the expression of the target protein. It has been shown that when truncated cold-shock protein (CSpA) mRNA is expressed under cold-shock conditions (cooled from WC to 15°C and held for 36 h), the polyribosomes are occupied for translation of the truncated c-subunit A gene, and no HI-type ribosomes can be formed for the translation of non-cold-shock proteins (JiangetaL 1996). As a result, only genes in the mRNA are translated, and expression of endogenous proteins is inhibited. There is a cloning region after the promoter in the pCold plasmid for insertion of the target gene, the expression of the target protein can be blocked using 1 mmol/L IPTG, and overexpression of the target gene can be induced by cold-shock at 15°C. Exogenous proteins expressed by this technique can often be left unpurified due to the extremely low expression of host background proteins, which greatly cuts down on the cost of production. For more product details, please visit Aladdin Scientific website.
