Site-specific nucleic acid endonucleases for protein engineering experiments
Site-specific nucleic acid endonucleases for protein engineering experiments
Site-specific nucleic acid endonucleases are involved in many aspects of nucleic acid biochemistry. Restriction enzymes and related enzymes have become the paradigm for enzymes that act on DNA. Countless efforts have been invested in trying to alter their specificity through rational protein design. The source for this experiment is "A Laboratory Guide to Modern Protein Engineering" [German] K.M. Arndt, K.M. Miller, eds.
Operation method
Protein engineering by site-specific nucleic acid endonucleases
Materials and Instruments
pMQ402 Move 3. Methodology For more product details, please visit Aladdin Scientific website.
Arabinose Phenylmethylsulfonyl fluoride Ni-NTA Agarose Binding buffer Wash buffer Elution buffer Dialysis buffer
Ultrasonic Breaker Cryo-Centrifuge
The methods listed here summarize amino acid site selection for targeted mutagenesis (see 3.1), MutH protein mutagenesis for expression in bacterial cells (see 3.2), and identification of MutH variants (see 3.3).
3.1 Amino acid selection for targeted mutagenesis
The sequences of the MutH protein and associated restriction endonucleases were obtained from the National Center for Biotechnology Information Basic Local Alignment Search Tool (BLAST) page (http : //www. ncbi. nlriLnih. gov /BLAST/) [ 24 ]. Additional sequences were obtained from the PSI BLAST server [ 25 ]. Sequences were aligned using the PAM250 matrix with the ClustalX program. The aligned sequences were analyzed using the GeneDoc program [27]. The program can be accessed by using the "Help" function within the program. The coordinates of the structures were obtained from the PDB database (http : //www. rcsb . org/pdb/ ). org/pdb/ ). The programs RasWin and Swiss PDB viewer are used for structure observation.
3.1.1 Identification of conserved residues related to DNA binding
After sequence comparison (e.g. with ClustalW or ClustalX), complete the following steps.
( 1 ) Enter the results of the comparison into the GeneDoc program (see 2.1; reference [27]).
( 2 ) Group the sequences using the "Group" function (e.g., one group contains MutH and the other contains restriction (nuclease).
( 3 ) Identify group-specific residues (see Note 1).
( 4 ) Use the "RasMol Script Dialog" function within GeneDoc to generate a RasMol script to map group-specific residues into (tagged in) the MutH protein structure.
( 5 ) Load the MutH structure into the RasMol program (see 2.1; Ref. [ 28 ] ), and execute the script exported from GeneDoc in the " RasMol Command Line" with the "Script" command. to observe group-specific residues.
( 6 ) Identify candidate residues located in what is assumed to be the DNA binding site.
3.1.2 Identifying residues that contact specific bases
( 1 ) Download existing sequences where restriction endonucleases form complexes with DNA substrates (or products).
( 2 ) Run Swiss PDB viewer to match the restriction endonuclease structure to the target MutH structure, using the backbone atoms of the 3 catalytic residues (e.g., D70, E77, and K79 of MutH) as seeds.
( 3 ) Enhance the fitting effect with the "Improve fit " function.
( 4 ) Output the coordinates of the overlapping structures to a spreadsheet program.
( 5 ) Calculate the distance from any atom of the target protein (e.g. MutH ) to the base in the overlapping structure that corresponds to the target base in the target protein.
( 6 ) Repeat these steps for all overlapping structures.
( 7 ) Calculate the average distance and identify the residue that is closest to the base of interest.
( 8 ) Compare the results with group-specific residue analysis.
( 9 ) Select promising residues for the targeted mutation.
3.2 Targeted Mutations of MutH Variants
MutH variants were cloned using the QuikChange procedure (Stratagen) modified as described by Kirsch and Joly [29], with plasmid pMQ402 (from Dr. M.G., University of Massachusetts Medical School, MA) as a template, and two oligodeoxynucleotides for mutation to suit the length of the resulting PCs. The plasmid pMQ402 (from Dr. M.G. University of Massachusetts Medical School, MA) was used as a template, and two oligodeoxynucleotides were used for mutagenesis to produce PCR products suitable for use at lengths of 50-500 bp (see Note 2).
3.3 Purification and characterization of MutH variants
To be suitable for in vitro testing, MutH variants must be purified.
3.3.1 Purification of MutH variants from bacterial cells
( 1 ) Transform XL-1 Blue MRF cells with a bacterial plasmid using standard molecular biology methods.
( 2 ) Cells are spread on LB culture plates containing ampicillin and incubated at 37°C overnight.
( 3 ) A single colony was selected and grown at 37°C in 500 ml of LB culture medium containing 75 μg/ml ampicillin.
( 4 ) Induction with 0.2% (m/V) arabinose (final concentration) at an optical density of 0.8 at 600 nm for 2.5 h at 28°C (see Note 3).
( 5 ) Centrifuge cells at 3000 g for 10 min.
( 6 ) Resuspend the cell pellet in 10 ml of binding buffer [see 2.3 (9)].
( 7 ) Break the suspension by ultrasonication 5 times for 1 min each time using a Branson ultrasonator with an output stage of 5 and a duty cycle of 50%, and cool the solution for 1 min between each ultrasonication.
( 8 ) Centrifuge the cell debris at 30,000 g for 30 min on a refrigerated centrifuge.
( 9 ) Gently mix the supernatant with Ni-NTA agarose syrup at 4°C for 30 min.
( 10 ) Transfer the Ni-NTA agarose to an empty column.
( 11 ) Rinse the column with 20 ml of Wash Buffer [see 2.3 (10)].
( 12 ) Elute with 0.5 ml elution buffer [see 2.3 (11)]. It is not necessary to remove the His tag as it does not interfere with endonuclease activity (see Note 4).
( 13 ) Collect each of the combined proteins using the optical density value at 280 nm as a judgment.
( 14 ) Dialyze the samples with 500 ml of dialysis buffer [see 2.3 ( 12 )] for at least 2 h at 4°C. Change the buffer twice.
( 15 ) Dialyze the sample with 500 ml of Dialysis Buffer G [see 2.3 ( 13 )].
(16) Dilute the sample 1:10 in dialysis buffer [see 2.3 ( 12)] and measure the light absorption at 280 nm to calculate the molar concentration of MutH using the theoretical extinction coefficient [30].
( 17 ) Store proteins at -20°C.
3.3.2 MutH cleavage analysis
Cleavage analysis of MutH and MutH variants is carried out using DNA substrates (oligonucleotides synthesized by methods described elsewhere, or PCR products, references [ 19 ] and [ 20 ]) containing unmethylated, hemi-methylated, or hypermethylated single d (GATC) sites (Figure 7.3). The upper and lower strands of the substrate were labeled with different fluorescent dyes (FAM and TET, respectively) to detect the cleavage on each strand. 
( 1 ) The 10 nmol/L DNA substrate is held in 10 μl of test buffer (see Note 1; appears to be Section 2.4, Article 1, in error) containing 500 nmol/L MutL and 10-500 nmol/L MutH (see Note 5).
( 2 ) The reaction mixture is incubated at 37°C.
( 3 ) Dispense the reaction mixture at appropriate intervals (10 s to 30 min), with 25 fmol of PCR product per portion, mixing thoroughly with 12 μl of template inhibitor (Perkin- Elmer) and 0.5 μl of Gene-Scan-500 TAMRA size standard (Perkin- Elmer).
( 4 ) Heat to 95°C for 2 min and cool immediately on ice.
( 5 ) Analyze samples on an ABI PRISM 310 Genetic Analyzer (Perkin- Elmer) equipped with a 47 cm (50 μm ID) capillary containing POP-4 polymer (Perkin- Elmer) spiked with 8 mol/L urea.
( 6 ) The samples were electrically injected into the capillary in 5 s and the run was completed in 30 min at 15000 V and 60°C using 1X Gene Analysis Buffer plus 1 mmol/L EDTA (Oerkin- Elmer) as electrode buffer.
( 7 ) Record the amount of cut and uncut fluorescently labeled DNA (Fig. 7.3).
( 8 ) Determine the cutting speed.