Parallel method experiments for expression and purification
Parallel method experiments for expression and purification
Proteins are diverse in nature, which makes it challenging to express and purify proteins using parallel methods. Parallel methods can especially be applied when multiple derivatives of a target protein are needed, as well as when multiple homologs are required. Typical protocols include evaluation of the target, cloning and mutagenesis of the target, expression screening, large-scale expression and purification, and analytical and biophysical testing of the resulting protein. This chapter describes some of the strategies and methods used for parallel protein expression and purification.
Authors: Burgess et al, Translator: Chen Wei, This experiment is from "Protein Purification Guide".
Operation method
Parallel method experiments for expression and purification Move I. Introduction Parallel protein purification is mainly a logistic challenge. There are many purification methods available for protein separation. Some methods inherently stand up better to the scrutiny of parallel methods than others (Gmslund etal., 2008a; Kimetal., 2004; Lesley et aL, 2002b). Most parallel purification strategies use purification labels, which can make simple standardized affinity purification methods easier. Although a method for cutting H i s tags with proteases is described in this scheme, other tagged or even tagless methods can be developed into parallel processes and are suitable for many applications. This paper is not intended to be exhaustive, but is intended to present the methods currently used in the authors' laboratories, and to serve as a discussion to elucidate the main aspects of parallel methods. To date the method has been applied to thousands of proteins and has provided proteins suitable for many applications. Successful protein expression and purification often requires the evaluation of multiple expression constructs . Many repeat truncation mutants and some homologous proteins require the identification of a suitable construct for recombinant expression. The ability to construct and characterize these mutants requires the ability to perform parallel expression screening and protein identification, which is a significant barrier to success. The definition of 'success' here is highly dependent on the end-use of the protein. When applied as a simple antigen, expression of a sufficient amount of the protein (even in aggregated form) for immunization is sufficient to claim 'success'. More often, biochemical studies or structural applications of proteins require not only that the purified protein be obtained, but also that the protein be folded correctly, contain appropriate cofactors or post-translational modifications, and be predominantly in its natural homogeneous state. Before defining any purification strategy or evaluation process, it is important to fully understand the end use and design steps to achieve the desired parameters. Crystallography is one of the most demanding end-use applications. Typically, proteins suitable for crystallization are also sufficient for biochemistry, immunology, and other functional research applications. These proteins should be properly folded (not only soluble), non-polymerized, homogeneous, have fewer unstructured regions, and be expressed at a level sufficient for multiple crystallization experiments. In most cases, screening of multiple target protein derivatives and proteins related to the target protein is required to meet these criteria (Fig. 41.1). These purified proteins require parallel processing and parallel characterization of their properties. Any experiments on proteins of interest should be preceded by bioinformatics evaluation. With the large amount of genomic information available, comparison of proteins that have little or almost no functional information can provide useful ideas for design guidance of protein expression. Software changes so rapidly that detailed methods of analysis are not provided here, but rather some guidance that can be used as a general approach.a Thus, the first task is to identify relevant proteins and compare them to identify their conserved regions. These regions typically constitute core structural domains that need to be conserved in expression constructs and can be used to characterize the boundaries of structural domains. The Basic Local Alignment Search Tool (B L A ST) (A ltsch u l e t a l . 1990) is the most commonly used tool for simple examination of related sequences. NCBI (h ttp ://blast.ncbi.nlm .nih.gov/B la s tc g i? CM D= W eb& P A G E _T Y P E = B lastH om e) provides multiple versions of this basic tool , which can query most genome sequences and can be used. Hidden M arkovm odel (H M M ) can be developed for more complex searches, although a simple PSI-B L A S T query is usually sufficient to identify the most useful and closely related sequences. Most proteins have been compared and categorized into families. Pfam (h ttp ://pfam.sanger.ac.uk/), a database of such comparisons, provides a wealth of useful information on comparative studies of related proteins, including structural domain boundaries and sequence conservation. U n iP ro t (h ttp :/,'w w w.uniprot.org/) (C onsortium , 2008) is likewise a useful source of summarized data, including predictions of secondary structure domains for most proteins. Ligands are also very useful tools for protein stabilization and activity detection. Ligand predictions are apparent from the annotations, but databases such as KEGGCh ttp://W W W W .genome.jp/kegg/) (K anehisa et al., 2008) and BioCyc (h ttp ://w w w w.biocyc.o r g ) (K arp et al., 2005) can provide eve ry ligand recommendations. Finally, a simple literature search can often reveal relevant proteins previously expressed by others to guide expression. While this may seem like a simple task, the rush to obtain constructs that can be expressed in systems already available in the laboratory often leads to hasty action without searching the previous literature. There are many other useful and easily accessible bioinformatics tools. The basic advice is to know what you want to Once useful information about a protein of interest has been gathered together, it is possible to design its expression construct. The most successful approach is not only to evaluate the full-length open reading frame (O R F ), but also to construct and test multiple truncated mutants of the O R F in parallel. Defining the N- and C-terminal boundaries is essential. We have observed many instances where a difference of one or a few amino acids at the end of a protein will completely change the protein behavior. Even if sequence conservation information is known, multiple N- and C-termini should be attempted. (1) Use B L A S T to query the existing genomic sequence of the protein in question and perform sequence comparisons. Tools such as P fa m can provide a convenient discussion of these queries and provide prevetted alignm ent and structural domain boundaries. Merge information obtained from literature searches. (2) Identify N- and C-terminal conserved sequence positions as initial truncation boundaries. In some cases, internal structural domain boundaries may be better. For example, kinase researchers usually study the catalytic domain itself. (3) Starting from these initial boundaries, select 5 to 10 additional N- and C-termini. The final set of expression constructs should be a matrix combination of these ends. We have found that adding about 4 amino acids provides a useful range of ends without increasing the number of constructs screened. An appropriate order of magnitude for screening full-length and empty vector controls is an I O X 9 matrix combination (90 constructs). (4) Check the selection of boundaries using end use as an indicator that critical regions have been retained. There are many different methods available for cloning and mutation. Each laboratory has its own preferences based on past experience . However, constructing a large number of unique expression clones is a daunting task. Some cloning methods are inherently more suited to parallel method processes. One common approach is to move Q R F into different receptor vectors using lambda In t/X is/fflF recombination at the att site (Hatley e t a l . 2000). Gateway ® is a commercially available form of this method and is widely used to construct expression vectors with different tags, but usually expresses additional amino acids encoded by the recombination site. T vectors (Harrison et al., 1994), topoisomerase-conjugated vectors (Shuman, 1994), and cre-Iox recombinant vectors (Liu et al., W 98) are also suitable for parallel cloning projects, but are not particularly flexible, and the construction of a customized vector is usually labor intensive. Our laboratory uses two methods, ligation-independent-cloning (LIC) (Aslanidis and de Jong, 1990) and polymerase incomplete primer extension (PIPE). extension (PIPE) (Klock et a l., 2008) have constructed thousands of expression clones . Both methods provide enough flexibility to clone ORFs into expression vectors, using conventional primers, quickly and with minimal reagents. The PIPE method will be described here, which also provides a convenient procedure for constructing truncated mutants. The PIPE method is based on PCR amplification to produce a mixture of incomplete extension products that contain varying degrees of unpaired 5' ends. This is similar to LIC in that a proofreading polymerase is used to remove the 3' end of the nucleotide. The unpaired 5' ends of the PCR products are derived from synthesized amplification primers. Therefore, the design of the oligonucleotide determines the 5' end of the cantilever and can be used in cloning strategies. A PCR product with approximately 15 complementary bases at the 5' end is prepared and inserted into the fragment and vector, which are annealed and transformed to give a stable expression plasmid (Fig. 41.2). Resuscitation. Take another 100 uL or 4 0 uL of resuscitated sensory cells and add to each well of a selective agar plate with glass beads. Holding the plate in your hand, gently shake it so that the glass beads move evenly over the entire surface of the wells. Turn the plate over, pour the glass beads onto the lid and discard. Incubate the plate upside down at 37°C for 12 to 16 h. Isolated clones can be screened by traditional methods, such as the isolation assay. Alternatively, a large number of clones can be screened by diagnostic PCR (dPCR) or SYBR-PCR. (1) Take 200 ul of LB liquid medium containing appropriate antibiotics into the wells of a flat-bottomed 9 6-well plate. Aseptically, select and transfer 1~4 isolated clones per transformation into a single well of a 96-well plate. Incubate the 96-well plate at 37℃, 250 r/min for 3 to 12 h. (2) Take 3 uL of each culture into a 96-well PCR plate. Return the rest of the culture to the shaker and continue to incubate for further growth for future glycerol conservation. (3) Add 47 PCR reagent mix, one gene-specific primer, and one vector-specific primer to each well containing organisms on the PCR plate. The primers should be designed so that they are amplified only if the vector contains the target fragment.D NA fragments are amplified for 30 cycles under conditions suitable for the primers. (4) Add 50 uL of 10 mmol/L ED TA to a flat bottom 96-well plate. Transfer 5 uL of each PCR product to each well and add 150 uL of SYBR buffer [40 ul 10 000 X SYBR Green I Dye (Sigma) diluted in 20 ml of 50 mmol/L Tris-HCl to make a 20x solution]. (5) The fluorescence value of each sample was determined using a fluorescence plate reader using the unamplified sample as a negative control. Excitation wavelength: 485 nm; emission wavelength: 525 nm. (6) S Y B R results are determined based on a relative range of positive wells having fluorescence values more than 4 times higher than those of negative or control wells . d P C R positive reactions can be confirmed by gel analysis or direct sequencing steps. Evaluation of clones on a small scale is an integral part of the clone selection process. Obtaining high yields of recombinant proteins that are soluble fractions of lysates is often the first major challenge encountered. However , many times this single criterion dominates the decision process, creating a false sense of success and leading to more work in subsequent stages. Screening methods such as fusion reporting genes (Lesley et al., 2002a ; W a W o et al., 1999) and clone imprinting (MartinezMolina et al., 2008) provide the means to screen thousands of protein derivatives. Fusion with molecular chaperones can improve solubility (Kapust and W a u g h , 1999), another strategy that has been used to define suitable construct boundaries for solubility. In many cases, these soluble proteins are aggregates of partially folded proteins mixed with heterogeneous Small-scale expression screens employ deep-well microtiter plates as a convenient way to propagate enough organisms to assess protein expression. Maximum growth of the organisms requires optimized expression conditions. In particular, the dissolved oxygen of the culture is the limiting factor for growth, so a short-throw plate shaker and a gas permeable plate lid can improve the final yield. Care must be taken at every step to minimize volume loss, as even small volume changes can have a very significant effect on final yield. (1) Prepare the desired clones for overnight incubation in a 9 6-well deep-well plate containing 8 50 0 ul of T B containing 2 % glycerol and the desired antibiotic. (8) Wet the 9 6-well filter plate with 300 uL of equilibration buffer, ensuring sufficient vacuum to leave a thin layer of liquid under the filter. Add the resin that has bound protein in the previous step and allow the supernatant to flow through the filter plate. Add 700 uL of rinse buffer [50 m mol/L H E PE S (pH 8.0), 300 m mol/L NaCU 40 m mol/L imidazole, 1 0 % glycerol, I m m ol/L T C E P] to each well and allow to flow.200 Centrifuge the filter plate for I m in to remove the remaining rinse buffer. Place a collection disk under the filter plate. Add 150 mL of elution buffer [50 m m ol/L H E PE S (p H 8. 0), 300 m m ol/L imidazole, 10 % glycerol, I m m ol/L T C E P] and incubate for 5 m in. Centrifuge the filter plate at 200 g for 5 m in to collect the purified protein. It is very important to avoid over-drying the resin in this step, as this may cause the resin bed to dry out, resulting in inadequate washing and elution of the resin itself. There are many methods for characterizing the quality and homogeneity of protein preparations. Determining the suitability of a particular expression construct or purification method requires recourse to the data obtained by these methods. Many of these tests have a high demand for proteins. However, in some cases, reduced methods can be applied to small-scale screens to provide useful parameters of protein properties. Some of the analytical methods listed in Table 41.1 can be applied to small amounts of protein. The production of soluble proteins is the first step in successful purification. In many cases, modification of the growth conditions, such as lowering the expression temperature or changing the expression construct (e.g., partnering with highly soluble molecules such as maltose-conjugated proteins), will greatly increase the solubility of the target protein product. However, these proteins often form soluble aggregates of reduced activity and stability relative to their correctly folded products. It is best to recognize and avoid these problems as early as possible. Analytical size exclusion chromatography (ANSEC) is a convenient and predictive method that can be applied in parallel testing modes. By reducing the column size and increasing the flow rate, the screening method can achieve throughputs of 13 to 18 m i n per sample. By combining this method with the HPC autosampler, a full plate is obtained from the small-scale method. (1) 用 20 m m o l / L Tris ( p H 7 . (1) Use 20 m m o l / L Tris (p H 7.5), 200 m m o l / L NACL, 0.25 m m o V LTCEP, 3 m m o l / L NAN3 to balance the appropriate size exclusion column (size exclusion column, Shodex 8 X 300 m mProtein K W -803 column with 6 X 50 m m Protein K W -G guard column). guard column). The flow rate is I.0 ~ I.5 mL/m i n . About 30 m i n is required. (2) After the column is properly balanced and the A 280 nm baseline is stabilized, inject 5 to 15 uL of sample protein (depending on the concentration). The sample should be injected before the sample and after approximately 50 samples (volume) with a suitable protein molecular mass standard and a blank buffer. (3) Figures 41.3 A and B show a comparison of typical results. Although high resolution protein size determination is not possible with fast flow rates and relatively short columns, the relative proportions of the high molecular weight aggregates and the individual states of the proteins can be easily seen in the graphs. In general, a consistent monomorphic state of protein is preferred over protein contaminated by protein aggregates, even if the initial purification yield is small. In order to choose the correct one among the many constructs, it is necessary to understand the ultimate use of the protein. For example, protein crystallization requires that the protein be unidispersed, homogeneous, and free of nonconstitutive regions. Although many constructs may provide sufficient soluble protein for crystallization, evaluation of SDS-PAGE, ANSEC, ligand binding and protease stability can effectively narrow the selection to tilted candidates. The more information available at the small-scale screening stage, the better the selection decision will be. Large-scale parallel expression purification of proteins usually requires special equipment. The scale of purification should be appropriate for the end use of the protein. IL bacterial expressions are usually sufficient to meet the needs of most laboratories. In most cases, the simple use of several L shakers is sufficient, but this method is difficult to apply when enlarged by a factor of 10 or 100. The airlift fermentor (http://w w w .gnfsystems.c o m ) provides a convenient means of expressing 96 different proteins on a scale comparable to that of the conventional IL shaker (Fig. 41.4) (Lesle y, 2001). Bacterial cultures with ()D values of 25 to 30 can produce several grams of organisms for subsequent processing. Lysis and purification of multiple cultures is the greatest logical challenge in large-scale parallel processing. Customized automated operating systems facilitate this, but it is more often achieved by simplifying the lysis and purification methods. The method given here was used to purify protein with a Hiss label containing a TEV cut-off site to facilitate label removal. We have found that this method is reliable even without automation and is easy to parallel scale up to many proteins. Over 300 proteins are processed in our laboratory each year using this method. (1) Directly take fermented protein-expressing organisms and incubate at room temperature with 0.25 m g of ovalbumin per mL for 30 mIn, centrifuge to collect the precipitate, and freeze the precipitate until use. Thaw about 3 g of starch in 80 mL of lysis buffer [50 m m o l/L HEPES (pH 8.0), 50 m m o l/L NACL, 10 m m o l/L imidazole, Imidazole/Ltris (2-carboxyethyl) phosphine hydrochloride (TCEP)] for purification. For purification, each batch of bacterial starch was frozen and used for purification. Each bacterial starch was cleaved as follows. (2) Homogenize the bacterial precipitate with lysis buffer using a homogenizer (Omni International) at 35 000 r/m i n (maximum setting) for approximately 80 s. Each cycle consists of sonication of the probe near the bottom of the homogenate for 6 s, followed by sonication of the probe near the surface of the homogenate for 3 s. Both processes resuspend and efficiently lyses the organisms. Both processes resuspended and effectively lysed the organisms. If insufficient lysis is observed, the lysate can be allowed to pass through a microfluidizer (Microfluidics, Inc.) or a French press. (3) The lysate is centrifuged at 32,000 g for 30 m i n . The clarified lysate was prepared for affinity purification. In parallel purification, conventional purification methods can be a significant bottleneck. Instruments and procedures for using H P L C for multiple sample processing have been described (M c M u l l a n e t a l . , 2005). However, for most applications, optimized affinity purification can provide sufficient purity as well as the necessary throughput. We have found that two-step tin purification combined with protein hydrolysis to remove the purification tag is an excellent purification method and can be used in parallel in hundreds of samples at different scales. The method has been shown to provide protein purities suitable for crystallization experiments and enzymatic screening studies. Our vectors contain a T EV protease cleavage site followed by a HIs tag. This method is applicable to other tags and cleavage enzymes. (1) Pipet each portion of clarified lysate into two I.5 m L gravity-fed nickel chelating resin columns that have been pre-equilibrated with lysis buffer. Each effluent was collected in a waste tray. (3) Place each column on a PIM O desalting column pre-equilibrated with digestion buffer [20 m m ol/L H E PES (p H 8.0), 200 m m ol/L NaCl 40 m m ol/L imidazole, I m m ol/L T C E P]. Add 2. 5 mL of elution buffer to the nickel chelating resin and collect the eluate directly onto the PD-10 column. (4) Add an additional 3. 5 mL of Digestion Buffer to elute proteins from the PD 40 column and collect the eluate into a 15 mL disposable tube. A small sample is removed for protein detection and SDS-PA G E analysis. (5) The H i s label is next removed by protease hydrolysis. The protease itself carries the HIS tag. Thus, subsequent flow-through of the town resin removes the protease, proteins not cleaved by the protease, and proteins that are nonspecifically adhered to the nickel resin, and the target proteins are allowed to flow through the column. For every 15 m g of protein eluted from the P IM O column, I m g of H i s-labeled T EV protease is added (Tropea et aL , 2009). The total volume of digestion was made up to 9 m L with digestion buffer. Mix by turning at room temperature for 2 h or overnight at 4°C. (6) Pour each digested protein mixture into an I.5 m L gravity self-flowing nickel chelate column pre-equilibrated with Digestion Buffer. Each flow-through was collected into a 50 m L conical tube. After the digested mixture has passed through the column, an additional I.5 m L of Digestion Buffer is added to the resin to remove residual cleaved proteins and collected in the same tube as above. The resulting 10.5 m L elution volume contains the purified proteins with the H i s tags removed. For more product details, please visit Aladdin Scientific website.
express and the proteins that are closely related to it, and use this information to design your experiments.
mixtures (Fig. 41. 3A, B ). This can become problematic during purification, often resulting in low and unstable yields of the product and posing problems in obtaining specific highly active proteins or crystallization of the protein. Partial hydrolysis of proteins can be used to determine stabilization boundaries. The stable fragments obtained are identified by mass spectrometry and can be used to define subsequent expression constructs. The addition of a ligand for the target protein also has the effect of stabilizing it (Fig. 4 1 . 3 0 . Ligands can be identified by screening methods, such as Therm ofluor (Pantolianoetal., 2001), or predicted by annotation and experimentation. To the extent possible, additional protein analyses should be performed at an early stage, with the results of the analyses factored into the decision to select expression constructs for large-scale studies. With these caveats in mind, expression screening should be sufficient to allow implementation of these tests at scale.
A full plate obtained by the small-scale method can be screened for % purified protein in less than one day. The chromatographic method presented here was run at I 3.3 m in on an Agilent HPLO with a 384-well autosampler with chilling capability. An alternative method with a slightly higher resolution of 18 m in is available and is more gentle on very high molecular weight samples.
(2) Add 7. 5 m L of wash buffer [50 m m o l / L H E P E S ( p H 8. 0), 300 m m o l /L N a C l , 40 m m o l /L imidazole, 1 0 % glycerol, I _ 1 / L T C E P ]. Add 0.5 m L of elution buffer [20 m m o l / LH E P E S ( p H 8.0), 300 m m o l / L imidazole, 1 0 % glycerol, I m m o l / L T C E P ]. The volume of elution buffer should not exceed 1/3 of the volume of the resin bed, the purpose of which is to push the elution front of the buffer along the bottom of the resin to reduce the elution volume in the next step.