The field of protein engineering is primarily concerned with the most fundamental sequence determinants regarding the design of new enzyme activities or folds, as well as the understanding of the correct folding and stabilization of proteins. To date, a tremendous amount of effort has been devoted to the study of methods for designing and constructing peptide libraries. The most commonly used method is phage display technology used to screen for specific binding proteins with high affinity. The source of this experiment is "A Laboratory Guide to Modern Protein Engineering" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Synthesis of a parsimonious evolutionary library of the Ras-binding structural domain of the Raf protein and rapid screening of dihydrofolate reductase clones using the fragment complementation method
Materials and Instruments
Oligonucleotide primers Tag polymerase BL21 Electroreceptor cells Move 3.1 General For more product details, please visit Aladdin Scientific website.
Ampicillin Kanamycin
Agarose Gel
3.1.1 PCA Requirements for Spatial Alignment
The three-dimensional orientation of the PCA fragments is critical for the correct folding of the PCA reporter protein, depending on the orientation of the N- or C-terminus of the target protein forming the complex (Figure 15.1 illustrates the spatial position considerations in linkage design). Therefore, when designing a protein-pCA fragment fusion, it is important to link the protein fragments together in such a way that they can fold to form a natural structure. The ability of the fusion protein to fold correctly is influenced by both the terminal orientation of the fusion protein and the length of the inter-fragment head (linker). We have applied the fragment GGGGS to a number of different proteins and have shown that this sequence works well in E. coli, yeast and mammalian cell expression systems. We believe that this sequence improves the elasticity and solubility of fusion proteins, thus facilitating their assembly, and ensures the stability of fusion proteins due to the absence of natural protease recognition sites in this sequence. Although this type of junction is favored, it does not necessarily mean that using it will result in fragments that can be complemented [ 31, 32, 39]. Nevertheless, it is important to avoid the use of large hydrophobic and rigid amino acids such as proline and branching amino acids. In most protein engineering problems, the three-dimensional structure of the target proteins forming the complex is known, so the orientation of the complex can be designed and the length of the desired junction can be calculated. For DHFR PCA, the spatial conformational requirements of the N- and C-termini of the fusion protein are known [ 31, 44]. For example, if the target protein is fused to the C-terminus of F[ 1 , 2 ] and the N-terminus of F[ 3 ], the required junctions for the construction can be short or even unnecessary because the topology of the DHFR is already taken into account in this construction method; however, if the target proteins are constructed to the N-terminus of each of the two fragments, at least two amino acids have to be inserted between the two fusion proteins as junctions (each peptide bond) in order to enable the DNFR to be assembled correctly. However, if the target proteins were constructed separately into the N termini of the two fragments, for the DNFR to assemble correctly, at least two amino acids would have to be inserted between the two fusion proteins to act as a junction (approximately 3.75 A per peptide bond), since the distance between the N termini of the two DNFR fragments is nearly 10 A. This construction was chosen in the case of ras and RBD. However, looking at the structure of the complex between raf RBD and raplA, which is a similar protein to ras, we found that the C-terminal distance between the two proteins is 40 A, which requires the insertion of a junction of at least 6 amino acids in each fusion protein. For library screening, we set the total length of each junction at 14 amino acids, including restriction endonuclease sites, to ensure that the fusion proteins are flexible enough. 
3.1.2 Controls and rigor
Prior to utilizing the DHFR PCA for protein engineering studies and library screening, rigorous control experiments must be performed to estimate its sensitivity and stringency for the particular system being tested. Ideally, before performing library screening, the experimenter should have a rough idea of the limits of the dissociation constant Kd of the PCA for a given interactor. Different pairs of interacting proteins have different sensitivity limits (the maximum Kd that can be detected by PCA), but the Kd value is influenced by the level of expression of the fusion protein, the proportion of soluble proteins in the total amount of protein expression, as well as by the properties of the protein itself, such as stability, solubility, and protein folding and binding parameters. If PCA is so sensitive that it can detect very weak interactions between two specific proteins, it will not be able to select the best interacting protein pair among many clones; that is, the experiment is too sensitive and thus loses its rigor. Therefore, it is crucial to balance the relationship between sensitivity and rigor. To address this issue, it is often necessary to do some control experiments prior to PCA experiments, although not all of these controls are relevant to the particular protein engineering study, and we will return to this issue later.
( 1 ) Spurious reassembly: For PCA to work effectively, weak or non-specific interactions between fragments should be avoided. For a given interacting protein pair test system, the sensitivity at which PCA is effective can be estimated from controls 4 and 6. If the sensitivity is too high, as in the case of growing colonies that should not have interacting protein pairs (Fig. 15.2A), the sensitivity can be reduced by lowering the expression level or by a tight mutation as described in Control 2 (Fig. 15.2B).
( 2 ) Stringency mutant: the effect of mutating the side chains of amino acid residues on the interacting surfaces of the two segments of the DHFR, as has been reported for the I114A mutation of F[ 3]. In clones expressing protein pairs that interact by forming leucin zippers, the colony growth rate and number of colonies were unable to distinguish between the binding efficiencies of the different protein pairs. Later, the introduction of the mutation I114A in F[ 3] altered the sensitivity of PCA so that it could be applied to the leucine zipper system. This change in sensitivity can be measured by the selection factor of a one-step screen, which is equal to the number of co-transformed cells divided by the number of colonies that survived the selection pressure. The higher the value the higher the tightness, so that the best interacting leucine zipper heterodimers can be selected under competition with a reasonable number of replicates [32]. 
( 3 ) Fragment sw apping: Regardless of which fragment of the PCA system the two interacting proteins are fused to respectively, theoretically the interaction of the two target proteins should not be affected. Therefore, replacement of the fragments fused to the two target proteins should give similar and comparable results.
( 4 ) Noninteracting protein: If a protein is known not to interact with any of the target proteins used in the PCA test, no PCA response should be detected (Fig. 15.2A), and overexpression of this protein alone will not compete with the known interactions.
( 5 ) Titration and reduction of reporter protein activity by competition assay: The activity of the reporter protein should vary with the ratio of the two fusion proteins expressed; and simultaneous overexpression of either of the interacting proteins alone should attenuate the PCA response. However, it should be kept in mind that the corresponding solubility and stability of each fusion protein, the affinity of the interacting proteins compared to their intracellular concentration, and the sensitivity of the PCA all have an impact on the activity of the titrate reporter protein. Thus, it is possible to modulate the activity of the reporter proteins by decreasing the expression level of the fusion proteins or by integrating Control 2 and Control 6, thus reducing the complementation efficiency.
( 6 ) Disruption of interactions: It is predicted that insertion of a specific point mutation or deletion of a mutation in one of the monomers forming the complex that disrupts or attenuates the target-protein interactions will also affect the PCA response.
If the nature of the model used for the study in the protein engineering program is fairly well known, then only Control 1, Control 2, Control 4, and Control 6 are necessary to determine the specificity and accuracy of the experiment. complementary mutation experiments with RBD-ras and the effect of mutations on the affinity constant Kd have been reported, and based on these data we constructed a number of mutations that decreased the Kd value of the RBD-ras interaction by three orders of magnitude (Fig. 15) (see Fig. 15). reduced the Kd value of RBD-ras interaction by three orders of magnitude (Fig. 15.3 example). We used both these mutants and others in the DHFR-PCA test to ensure that this experiment could detect RBD-ras interactions at an order of magnitude of 1 μmol/L. In addition, published mutations that reduce protein stability, such as mutating hydrophobic amino acid residues in the core region (Val, Leu, or lle ) to Ala, can be used for stringency testing. 
3.2 Synthetic libraries
( 1 ) In order to obtain an unbiased library, a template is constructed by replacing the insertion region (variable region) of the gene with a stop codon, and inserting a frame shift and a specific restriction endonuclease site to ensure that it is unambiguously identified (see Note 1).
( 2 ) Two partially overlapping (typically 18-20 bp) PCR products are required to produce each library. For example, for PCR reaction 1, one primer is used that is complementary to the promoter region of the vector (120 bp upstream of the start codon, Figure 15.4) and the other primer is complementary to the 5' segment of the parsimonious target. For PCR reaction 2, a double-armed primer and a primer that hybridizes to F[1,2] (120 bp downstream of the readable frame 3', Figure 15.4) are required. Typically, the PCR reaction is programmed as follows: warm-up at 94°C for 1 min; then 25 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 30 s (see Note 2); and finally, extension at 72°C for 10 min to ensure complete extension.
( 3 ) PCR products were analyzed by agarose gel electrophoresis. PCR products were visualized on agarose gels using a Gelstar and a Dark reader (see Note 3), which uses blue light at 400-500 nm, a wavelength that is not damaging to DNA. If a target band is obtained, the remaining PCR product is used for gel cutting and recovery.
( 4 ) Purify the pelletized product using QiaexTMII (see Note 4).
( 5 ) Approximately 300 ng of product from PCR reactions 1 and 2 were mixed (Figure 15.4 and Note 5) and annealed to the 5' and 3' ends of PCR reactions 1 and 2, respectively, with the addition of 0.2 μmol/L of terminal primers (complementary to the promoter region and F [ 1, 2 ], respectively). 30s at 94°C, 30s at 52°C, and 30s at 72°C for a total of 10 cycles; finally, extend at 72°C for 10 min to ensure complete extension (see Note 6).
( 6 ) The PCR product and vector pQE-32∆F [ 1, 2 ] were digested using a suitable restriction endonuclease (in this case, SphI and XhoI) (see Note 7).
( 7 ) Purification of the digest was performed as in step (4). 
3.3 Library cloning and recovery
( 1 ) The ideal ratio of insert fragments to vector is (2 : 1 ) ~ (3 : 1 ). We limit the concentration of DNA used for ligation to 10 ng/μl using 1 mmol/L ATP at 16°C overnight (see Note 8 ).
( 2 ) The ligation reaction system is treated at 65°C to inactivate the enzyme, extracted with chloroform, then precipitated with ethanol, and the resulting DNA is allowed to air-dry for a few minutes and finally resuspended in 30 μl of deionized water.
( 3 ) On the same day, an E. coli cell line SS 320 (see Note 9) should be prepared in the electroreceptor state (see 15.3.8). The ligation product obtained in step (2) should be mixed with 300 μl of SS 320 cells and added to a 2 mm wide electro-transfer cup for electro-transformation using a GenepulserTMII. Set the instrument parameters as follows: 2.5 kV, 25 μF, 200~400 Ω. For best results, the time constant should be 3.8~4.5 for 200 Ω and 7.6~9.0 for 400 Ω. Immediately after pulsing, add 1 ml of pre-cooled SOC medium. Transfer the cells to a 15 ml conical tube, wash the spinning cup twice with SOC medium to maximize the recovery of spinning cells, and resuscitate the cells in 5 ml of dry SOC medium at 37°C for 30 min at appropriate speed.
( 4 ) Take an appropriate amount of cells, dilute 104 times, smear the plate, and count the colonies to measure the efficiency of ligation and cloning (see Note 10). The remaining cells are added to 250 ml of LB medium with appropriate additives [see 15.2.2 ( 9 )] and cultured for DNA extraction.
( 5 ) DNA is extracted using the Qiagen Midiprep Kit or other similar base lysis kits [47].
3.4 Library Screening
( 1 ) Using a 1 mm wide electrotransfer cup, electrotransform 100 ng of the library obtained in 15.3.3(5) into 65 μl of BL21 pREP4 cells already carrying the plasmid pQE-32 ras-F [3]. Instrument parameters: 1.25~1.6 kV, 25 μF, 200 Ω. Time parameters were set to 3.7~4.2 for GenepulserTM II and 4.0~4.6 for Electroporator 2510. Cells were resuscitated in SOC medium at 37°C for 30 min.
( 2 ) Cells were washed twice with PBS to remove the SOC medium.
( 3 ) Spread the cells onto selective plates as described in 15.2.3 ( 6 ) and incubate at 30°C for 24~72 h (see Note 12). At the same time, another portion of electrotransformed cells was removed and diluted 103-fold to coat the plates for counting and comparing with the positive control to estimate the transformation efficiency and cell viability of the library clones. For example, we simultaneously transformed the same amount of vectors expressing wild-type RBD and F [ 1, 2] fusion proteins, and diluted the electro-transformed cells 104~105 times to coat the plates. At each step of the procedure, all measurements were made to avoid cross-contamination of the library with the wild-type positive control.
3.5 Cloning competition experiments
Adapted from reference [32].
( 1 ) After appropriate time of incubation, recover cells from 15.3.4 ( 3 ) plates with a small amount of selection medium, add to 25 ml of selection medium and incubate at 30°C 250 r/min.
( 2 ) After 24 h of incubation, take 1 μl of saturated culture and dilute it into 2 ml of fresh selection medium.
( 3 ) Step (2) can be repeated until the library reaches the target abundance. Normally, after 12 generations (12 D), the library is largely concentrated. However, this varies from system to system and is influenced by factors such as library parsimony and the use of tight mutants (see 15.3.1.2).
( 4 ) At any step, a 104- to 105-fold dilution of the saturated culture can be applied to a selective plate to qualitatively test for competition efficiency. As successive competition proceeds, the size difference between colonies will gradually decrease and the average size of the colonies will increase.
( 5 ) In any generation, 10 μl of library can be removed and added to 2 ml LB ( 100 μg/ml ampicillin, 25 μg/ml kanamycin) for overnight incubation, and then the DNA of the clones in the library can be extracted using QIAprep ( see Note 14).
3.6 Isolation and sequencing of clones
The following procedure applies both to the extraction of plasmids from independent clones contained in the library and to the extraction of plasmid mixtures in the operations described above.
( 1 ) 300 ng of DNA was removed from the clone library and digested by adding restriction endonuclease. The restriction endonuclease sites used are present in the pREP4 and pQE-32 ras-F [ 3 ] plasmids, but not in the library vector. For this purpose, we used Xmal, EcoNI, and Xba l ( see Note 15).
( 2 ) Take 1/10 of the digest product, transform XL- 1 Blue receptor cells, and apply 20 μl to an LB plate containing 100 μg/ml ampicillin (see Note 15).
( 3 ) Pick the colonies and culture them in LB containing 100 μg/ml ampicillin.
( 4 ) Extract high quality DNA for sequencing. For 96 samples, use the MontageTMkit; for smaller samples, use the QIAprep column kit.
( 5 ) We use primers for sequencing that can only anneal to library plasmids (e.g., F [ 1, 2 ] internal sequences) (see Note 16 ).
( 6 ) Sequencing.
3.7 Protein purification and assay of its properties
Analyze the sequencing results and select the clones of interest to be aligned in the appropriate position in the 96-well plate. At this point, the clones can be transformed into XL-1 Blue cells and frozen as a backup.
( 1 ) Selected clones are reconstituted to fuse only with the 6X His tag and express the protein (see Note 17). The expression of the 6X His clone is detected by induction (see Note 18).
( 2 ) Extract the plasmid of the correctly expressed clone.
( 3 ) Transform BL21 pREP4 cells, spread on plates containing 100 μg/ml ampicillin and 25 μg/ml kanamycin, and incubate at 37°C overnight. If multiple clones are manipulated in parallel, a 24-well plate may be used. In this example, do not use more than 20 μl of receptor cells per transformation, and apply a maximum of 20 μl per plate; if the maximum permissible volume is exceeded, the cells will not be completely absorbed into the medium.
( 4 ) The next day, pick the colonies in 2.5 ml of LB containing appropriate antibiotics and incubate at 37°C overnight (see Note 19).
( 5 ) Add the saturated culture to 25 ml of TB medium containing the appropriate antibiotic at a ratio of 1:10 (see Note 20).
( 6 ) Add IPTG to a final concentration of 1 mmol/L and incubate at 37°C for 90-120 min. Harvest cells for direct protein extraction or store at -80°C (see Note 21).
( 7 ) Resuspend cells with 1 ml of Buffer A [ see 15.2.6 ( 8 ) ] and rearrange in a 96-well plate (see Note 22). centrifuge at 3200 g for 40 min to remove most of the insoluble fraction.
( 8 ) A 0.2 μm 96-well PVDF plate containing 200 μl of 50% Ni-NTA Superflow resin. The volume expander is mounted on top of a 0.25 mm 96-well glass-life fiber filter plate, which is then mounted on the sealing block of the vacuum manifold (see Note 23). Then, add 900 μl of sample to the first filter plate and 100 μl of ethanol to minimize the risk of cross-contamination. Filtration is carried out at a pressure of approximately 500 mbar until all the sample has passed through the plate (see Note 24).
( 9 ) The PVDF plate should now contain the resin and sample extract. Stop filtration, remove the PVDF plate from the collector, install it on the sealing block of the vacuum manifold, and then filter at approximately 100 mbar until all sample has passed through the resin (see Note 25).
( 10 ) Wash twice with 800 μl of Buffer B [see 15.2.6 (9)], setting the vacuum pressure to 500 mbar each time (see Note 26).
( 11 ) Place the collector and elute the sample 4 times with 100 μl Buffer E at 100 mbar [see 15.2.6 (10) and Note 26]. Add 1 mmol/L DTT and adjust the pH to 5 (see Note 27). At this point, the sample can be used directly for characterization (see Note 28).
3.8 Preparation of SS 320 and BL21 pREP4 pQE-32 ras-F [ 3 ] receptor cells
Prepare cells according to the method of reference [ 48 ].
( 1 ) Pick SS320 or BL21 pREP4 pQE-32 ras-F monoclones in 5 ml LB or 5 ml SOB medium and incubate at 37°C for 5 h to overnight.
( 2 ) Add 2.5 ml of culture to 500 ml LB or 500 ml SOB medium and incubate at 300 r/min at 37°C until the OD600 is 0.5~0.7.
( 3 ) Ice bath for 10~15 min and transfer to pre-cooled 1 L centrifuge flask.
( 4 ) Centrifuge at 5000 g for 20 min.
( 5 ) Discard the supernatant, resuspend the organisms with 5 ml of ice water, add 500 ml of ice water and mix well, and centrifuge as in step (4).
( 6 ) Immediately discard the supernatant and resuspend the organisms with the remaining liquid.
( 7 ) Add 500 ml of ice water and mix well, centrifuge as in step (4).
( 8 ) Discard the supernatant immediately and resuspend the organisms with the remaining liquid.
( 9 ) If the cells are to be used for electrotransformation, transfer the resuspended cells to a pre-cooled 50 ml centrifuge tube and centrifuge at 5000 g for 10 min at 2°C. Estimate the volume of the organisms (typically 500 ml of culture will yield 500 μl) and resuspend the cells in the same volume of pre-cooled water on ice. aliquot 50 to 300 μl of the cells into the pre-cooled centrifuge tube, the density of the cells will be approximately 2X1011 cells/ml. 1011 cells/ml.
( 10 ) To freeze the electrotransformed cells, add 40 ml of pre-cooled 10% glycerol to the cells obtained in step (8) and mix well. Centrifuge the cells as in step (9), estimate the volume of organisms, and resuspend the cells by adding the same volume of pre-cooled 10% glycerol on ice. aliquot 50-300 μl of cells into pre-cooled centrifuge tubes and freeze on dry ice, store at -80°C.
3.9 Preparation of XL-1 Blue and BL21 pREP4 Receptor Cells
Cells are prepared according to the method in Ref. 49 with the following modifications: cells are washed only once after the first centrifugation, and the centrifuge bottles are inverted on paper at 4°C to remove traces of medium (see Note 29).
( 1 ) Add overnight culture to 200 ml of SOB or LB medium and incubate at 18°C (200-250 r/min) until the OD600 is approximately 0.6.
( 2 ) Ice bath for 10 min and transfer to a 500 ml centrifuge flask.
( 3 ) Centrifuge at 2500 g at 4°C for 1 min.
( 4 ) Resuspend the organisms with 80 ml of pre-cooled transformation buffer and ice bath for 10 min, centrifuge as in (3 ).
( 5 ) Gently resuspend with 20 ml of pre-cooled transformation buffer and add DMSO to a final concentration of 7%.
( 6 ) Ice bath for 10 min.
( 7 ) Aliquot cell resuspension and freeze in liquid nitrogen.