Antibody simulation experiments based on the framework of fibronectin type III structural domains

Summary

We developed the use of the 10th fibronectin type III structural domain (FNfnlO) of human fibronectin as a framework to show the use of multiple surface chain sets for binding to other molecules. We refer to the FNfnlO variants with new binding functions as "monomers (orphans, monosomes)". The source for this experiment is "A Guide to Modern Protein Engineering Experiments" [German] K.M. Arndt, K.M. Miller, eds.

Operation method

Antibody simulation experiments based on the framework of fibronectin type III structural domains

Materials and Instruments

pAS38 pAS45 pAS47
Polyethylene Glycol (PEG) Sodium Chloride Solution HEPES Buffer Coating Solution Tris Buffered Saline TBS-Tween-20 Agarose-Phosphate Solution
LB SOC YT

Move

3.1 Physical repository construction

We used the Kunkel mutation technique to construct most of the physical libraries Although we demonstrated that the AB strand sleeve can be used to bind target molecules, in most cases we used the "upper" end of FNfn10 (BC, DE, and FG strand sleeve; Fig. 6.1A ) as the target molecule binding site [ 10]. Our method is based on Bio-Rad's Mutagene kit [17]. A physical library containing about ¹⁰⁹ independent clones can be easily prepared by electro-transformation with E. coli SS320.



3.1.1 Preparation of uracil single-stranded phage (for monovalent phage display and yeast two-hybrid screening)

( 1 ) Transform CJ236 with pAS47, pAS38 (for solo-gene III) or pYT 45 (for B42-solo) and select colonies in LB-AP plates.

( 2 ) Inoculate one colony into 2 ml 2X YT-Ap-Chloramphenicol medium and shake at 37°C overnight.

( 3 ) Fill 30 ml of pre-warmed 2X YT-Ap medium supplemented with 0.25 μg/ml uridine into a 250 ml specimen triangular flask with depression, inoculate with 300 μl of CJ236 overnight medium, and add ¹⁰¹⁰ cells/ml of auxotrophic phage KO7 (Promega). Grow at 37°C with vigorous shaking for 2 h. Add Km to reach a final concentration of 70 μg/ml. grow overnight at 37°C with vigorous shaking.

( 4 ) Centrifuge at 12000 g [10000 r/min with SS-34 (Sorvall) or equivalent] for 10 min at 4°C. Transfer the supernatant to a new tube and centrifuge again at 12000 g for 10 min. Transfer the supernatant to a new tube. Add 4.5 ml of PEG/NaCl solution and mix thoroughly. store at 4°C overnight.

( 5 ) Centrifuge at 17200 g (12000 r/min, SS-34 rotor) for 20 min at 4°C. Dispose of the supernatant and place the tube upside down on a stack of paper towels for 1 min. Remaining liquid was wiped off with a paper towel.

( 6 ) Suspend the phage precipitate in 1 ml TBS and transfer to a microcentrifuge tube. Centrifuge in a microcentrifuge at maximum speed for 2 min and transfer the supernatant to a new microcentrifuge tube. Add 150 μl of PEG/NaCl solution and mix thoroughly. Allow to stand on ice for 30 min.

( 7 ) Centrifuge at maximum speed for 10 min at 4°C. Discard the supernatant, centrifuge briefly, and remove all liquid by pipette. Suspend the precipitate in 1 ml TBS.

( 8 ) Determine the phage concentration using XL-1 Blue and CJ236. Mix 100 μl of fresh E. coli (600 nm optical density OD of 0.5~1.0) with 10 μl of serial diluted phage solution. Conserve at room temperature for 15 min and spread on LB-Ap plates. Conserve at 37°C overnight. Titration values with CJ236 should be ^1012~1014 phage/ml and should be ¹⁰⁴ greater than values with XL-1 Blue.

( 9 ) Prepare uracil single-stranded DNA using the Qiagen Single-Stranded DNA Preparation Kit.

3.1.2 Preparation of uracil single-stranded phage (for multivalent phage display vector, JCFN )

( 1 ) Transfect CJ236 with JCFN plasmid. after heat excitation, mix with 30 ml of preheated 2 X YT and 500 μl of fresh CJ236 ( _OD600 value 0.5~1.0 ), then shake at 37°C for 6 h. The plasmid was used for the preparation of uracil single chain phage.

( 2 ) Complete steps (4) to (9) in section 3.1.1. To determine the concentration, mix 3 ml LB and 0.7% agarose (melted and cooled to 45°C ), 10 μl serially diluted phage solution and 100 μl fresh E. coli. Mix well and flush immediately into LB plates. The plates were incubated overnight at 37°C and the titers were determined.

3.1.3 Preparation of mutant oligonucleotides

( 1 ) Design an oligonucleotide so that it contains a 5' or 3' complementary region of approximately 20 and 15 bases, respectively, depending on the GC content. We used the codes NNK and NNS for "hard" randomization, where N stands for an equimolar mix of all nucleotides; K for an equimolar mix of G and T; and S for an equimolar mix of G and C. We also used the codons NNK and NNS for "hard" randomization.

( 2 ) Acrylamide gel electrophoresis of approximately 10 μg of the crude oligonucleotide dissolved in water is performed, the band corresponding to the correct molecular mass is cut out, and the oligonucleotide is electrically eluted (see Note 3 ).

( 3 ) Mix 2 μg of purified oligonucleotide, 3 μl of T4 polynucleotide kinase buffer, 5 U of T4 polynucleotide kinase (New England Biolabs), and 1.5 μl of 10 mmol/L adenosine triphosphate to 30 μl of water. allow to stand at 37°C for 2 h, then at 65°C for 15 min. store solution at 1-20°C.

3.1.4 Double-stranded DNA synthesis

( 1 ) Mix approximately 1 μg of uracil single-stranded DNA, 2 μl of phosphorylated oligonucleotide (see 3.1.3), 1 μl of 10 X annealing buffer (Bio-Rad Metagene kit), and add water to a volume of 10 μl (see Note 4). Prepare a control reaction with an oligonucleotide-free reaction.

( 2 ) Using a polymerase chain reaction (PCR) instrument, hold the reaction at 70°C for 5 min, then cool to 30°C at a rate of 1°C per minute. place the tube on ice.

( 3 ) To the annealing mixture, add 1 μl of 10 X synthesis buffer (Bio-Rad Metagene kit), 1 μl of T4 DNA ligase and 1 μl of T4 DNA polymerase (New England Biolabs). The samples were held on ice and at room temperature for 5 min each, at 37°C for 30 min, at 75°C for 15 min, and then cooled to room temperature. 1 μl of sample was passed through agarose gel ( electrophoresis). Samples containing oligonucleotides will give a product with a higher molecular mass than that obtained from a control sample without nucleotides.

( 4 ) Place a drop of the mixture on a 0.025 μm dialysis membrane dish floating on water and dialyze the synthesized mixture for 1 h. Then recover the dialyzed mixture in a new microcentrifuge tube. Keep on ice until use.

3.1.5 Preparation of electroporated competent cells

( 1 ) Culture E. coli SS-320 in 350 ml of super broth containing Tc until _OD600 reaches 0.8.

( 2 ) Rapidly cool the culture solution in a triangular flask in an ice bath and place on ice for 15 min. transfer the culture solution to a centrifuge flask and centrifuge for 5 min at 3900 g [ 4700 r/min with a JA -10 (Beckman) or equivalent rotor ] at 0~2°C (see Note 5).

( 3 ) Skim off the supernatant and suspend the cells in 10 ml HEPES buffer by tapping the centrifuge tube on ice. Add 390 ml of HEPES buffer and stir to suspend the cells. Centrifuge at 3900 g at 0~2°C for 5 min.

( 4 ) Skim off the supernatant. Suspend the cells in 10 ml of HEPES buffer and transfer the suspension to a 50 ml centrifuge tube. Rinse the first bottle with buffer and restore the cell suspension in a 50 ml tube.

( 5 ) Centrifuge at 4300 g (6000 r/min with SS-34 rotor) for 10 min at 0~2°C. Remove supernatant. Tap the tube on ice to suspend the cells in 10 ml of 10% glycerol, then add 20 ml of 10% glycerol and invert the tube several times to mix the suspension.

( 6 ) Centrifuge at 4300 g for 10 min at 0-2°C. Remove supernatant. Add 300 μl of 10% glycerol and tap the tube on ice to suspend the cells. Check the volume and add 10% glycerol to a final volume of 700 μl. Aliquot the suspension into two centrifuge tubes (350 μl each).

3.1.6 Phage DNA transfection by electroporation

( 1 ) Add cooled DNA [see 3.1.4, step (4)] to 350 μl of electroconverted susceptible cells in a microcentrifuge tube and keep on ice for 5 min. transfer the mixture to a pre-cooled electroporation tube (2 mm gap).

( 2 ) Electroporate the cells, e.g., in 2.5 kV high voltage mode using the BTX ECM395 Electroporator.

( 3 ) Immediately add 1 ml SOC to the tube and suspend the cells. Transfer the cells to a 250 ml trivet. Add 1 ml of SOC to the tube, wash the remaining cells and transfer to a trivet. Repeat this procedure 3 more times.

( 4 ) Add 20 ml of SOC to the trivets. shake the trivets at 180 r/min for 30 min at 37°C. check the concentration by placing a partially diluted suspension on an LB-Ap plate.

( 5 ) Add the entire suspension to 500 ml of preheated 2 X YT-Ap. For yeast two-hybrid plasmid preparation, shake overnight at 37°C and prepare the plasmid. For phage preparation, add ¹⁰¹⁰ pfu/ml helper phage KO7, shake overnight at 37°C and prepare phage as described in section 3.1.1.

For phage DNA transformation, after step (3), add 1.5 ml of mid-log phase SS-320 cells and transfer the suspension to a pre-warmed 120 ml 2 X YT-Tc. Determine phage concentration as described in section 3.1.2. incubate at 37°C for 4 h. Prepare phage as described in section 3.1.1.

3.1.7 Yeast library construction

For the yeast two-hybrid screen, a vector library was constructed in E. coli as described in section 3.1 and then introduced into yeast. The yeast transformation line is based on Gietz's method [18]. With this method, we obtained about ¹⁰⁶ independent clones in a typical case.

( 1 ) Grow yeast EGY48 overnight at 30°C in 20 ml YPD with shaking. add the culture to pre-warmed 300 ml YPD to give an _OD600 value of 0.25. allow the cells to grow until the _OD600 value approaches 1.

( 2 ) Centrifuge at 3600 g ( 4500 r/min, JA-10 rotor) for 5 min at room temperature. Skim off the supernatant and suspend the cells in 300 ml of sterilized water. Repeat centrifugation and suspension. Centrifuge again, suspend cells in 25 ml of sterilized water and transfer to a 50 ml centrifuge tube. Centrifuge at 3000 g (5000 r/min, SS34 rotor) for 5 min at room temperature. skim off the supernatant and suspend the cells in 1.2 ml of sterilized water.

( 3 ) Set 35 μl of the cell suspension aside to be used as a negative control. To the remaining cell suspension, add 7.2 ml of 50% PEG3350, 1.08 ml of 1 mol of lithium acetate, 500 μl of carrier single-stranded DNA (Origene), 45 μg of DNA and 900 μl of water. Mix thoroughly and dispense 360 μl each into 30 microcentrifuge tubes. Add 1/30 of the above materials except DNA (i.e. PEG, lithium acetate and carrier DNA) to the negative control tube and mix thoroughly.

( 4 ) Allow to stand at 42°C for 40 min. Centrifuge at 10,000 r/min for 10 s at room temperature in a microcentrifuge and skim off the supernatant. Suspend the cells in each tube in 150 μl of water. Cells were spread on a 15 cm diameter screening plate (YC Glc trp - ). 30°C for 3 d. The cells were stored in a microfuge for 10 s at room temperature at 10000 r/min.

( 5 ) Add 15 ml of water to each dish and scrape all plaques with a sterilized coverslip. Suspend all cells in a 500 ml centrifuge tube and centrifuge at 3900 g (4700 r/min, JA-10 rotor) for 5 min. Skim the supernatant and suspend the cells in 500 ml of water. Centrifuge again and skim the supernatant. Suspend the cells in 50 ml of water and measure the _OD600 to determine the cell density (1 _OD600 corresponds to approximately ^2X107 cells/ml). Add glycerol to a final concentration of 15% (V/V). Dispense and store cell suspension at -80°C.

3.2 Classification of Phage Display Banks

3.2.1 Panning

( 1 ) Add 50 μl of impregnation solution to the wells (1 μg of ligand per well) of an ELISA ELISA plate (Nunc Maxisorp, separable wells, C-bottom). Place the plates in a sealed box lined with a damp paper towel. Keep the box at 4°C overnight, or 37°C for 1 h.

( 2 ) Remove the liquid with a pipette and wash the wells twice with water. Add 360 μl of TBS containing 3% BSA to the wells and incubate at 37°C for 1 h. For the next round of experiments, prepare a control well; add ligand-free buffer and seal the well with BSA.

( 3 ) Remove the sealing solution from the wells and wash them twice with water. Add 12 μl of 5% BSA solution and 50 μl of phage suspension ( ^1011/well ). Allow to incubate for 2 h at room temperature.

( 4 ) Remove the phage solution and add 360 μl of TBST. pipette vigorously up and down. Wait for 5 min and then remove the solution (once in the first round and 5 times in each subsequent round).

( 5 ) Add 50 μl of TBST solution containing 10 μmol/L ligand to the wells. incubate at 37°C for 1 h. Stir vigorously up and down with a pipette to recover the eluate. Alternatively, 50 μl of acid elution buffer can be added to the wells and allowed to stand at room temperature for 10 min, stirring vigorously up and down with a pipette. Recover the second eluate into a microcentrifuge tube containing 3 μl of 2 mol/L Tris-base for neutralization.

( 6 ) Mix 5 ml of fresh XL-1 Blue ( _OD600 value of 0.5~1; grown in LB-Tc) and eluted phage. To the phage, add 100 ml of 2 X YT [ and isopropylthiogalactopyranoside (IPTG) up to 1 mmol/L to get a high yield of monomers ] and incubate at 37°C for 6 h while shaking vigorously ( longer incubation may result in the deletion of the monomer gene). Concentrations were determined as described in section 3.1.2 immediately after addition of 2 X YT. For phage, mix 5 ml of fresh XL-1 Blue and eluted phage, incubate at 37°C for 20 min, add 100 ml of 2 X YT (and IPTG, if desired), 100 μg/ml AP, and ¹⁰¹⁰ auxin phage KO7/ml; incubate at 37°C for 2 h with vigorous shaking, Km to a final concentration of 70 μg/ml and incubate overnight. Km to a final concentration of 70 μg/ml and incubate overnight. Immediately after 20 min of incubation, the concentration was determined as described in section 3.1.1. Isolate the phage (or phages) as described in sections 3.1.1 and 3.1.2. Repeat the entire procedure until the number of phages (or phages) eluted increases.


3.2.2 Identification of single phage clones

( 1 ) Prepare phage (or phage) from single clones. For phage, grow a single colony on 2 ml LB-Ap overnight. Mix 20 μl of culture medium, 2 ml LB-Ap (and IPTG, if desired) and ¹⁰¹⁰ phages/ml of helper phage KO7. Conserve overnight at 37°C with vigorous shaking. For phage clones, touch a single plaque with a sterilized toothpick. Place the toothpick in 2 ml of LB-Ap (and IPTG, if desired) containing 100 μl of fresh XL-1 Blue and incubate at 37°C with vigorous shaking for 6 h. Prepare phage (or phages) as described in sections 3.1.1 and 3.1.2.

( 2 ) For each clone to be tested, prepare a well with immobilized target molecules by completing steps (1) and (2) of Section 3.2.1. Prepare another control well by coating it with a coating solution that does not contain the target molecule (see Note 6 ).

( 3 ) Add 5 μl of 5% BSA solution and 50 μl of phage suspension from monoclonal preparation to the ligand-coated and control wells. Depending on the strength of the interaction, adjust the phage concentration (typical: ^108-1011 phages per well). Hold at room temperature for 2 h on a stirrer.

( 4 ) Rinse 5 times with TBST. Add TBST to all wells with a water spray bottle, then remove the liquid and pat the plate face down on a stack of paper towels.

( 5 ) To each well, add 50 μl of anti-phage-horseradish peroxidase antibody solution (Phamacia; diluted 1:2000 in TBST:BSA), and hold on a stirrer at room temperature for 1 h. Wash 5 times with TBST.

( 6 ) Add 50 μl of 1step turbo-TMB (Pierce), wait about 10 min until the blue color appears, and terminate the reaction by adding 50 μl of 2 mol/L sulfuric acid (use a tip with a filter to protect the pipette). Measure _OD450 with a plate reader and select clones with high affinity for ligand-impregnated wells and low affinity for control wells for further testing (sequencing, affinity optimization, and protein production).

3.3 Yeast two-hybrid screening

3.3.1 Preparation of target ("bait") plasmids

The bait gene was cloned in pEG202 such that the bait was expressed in the same readable frame as LexA. It is important to confirm that the LexA- decoy does not activate the reporter gene itself and does not interact with wild-type FNfn10. This was easily tested using the interaction mating method [13, 19 ].

( 1 ) RFY206 was transformed with the LexA- decoy plasmid [ or pEG202 as a negative control and pBait (Origene) as a positive control ] and pSH18-34 (β-galactosidase reporter plasmid; Origene) and selected on YC Glc his- ura- plates. EGY48 was also transformed with pYesTrp2 ( activator B42 plasmid; Invitrogen), pTarget (positive control; Origene), or pYT45 and selected on YC Glc trp-plates.

( 2 ) Parallel scratches were made on each of the two plaques on YC Glc his-ura- plates with LexA plasmid and reporter plasmid. The YC Glc trp - plates were scratched with the B42 plasmid in the same manner. both plates were conserved at 30°C for 1~2 d. The plates were then incubated at 30°C for 1~2 days.

( 3 ) Duplicate the two plates to one YPD plate. Scratches from both plates should be perpendicular to each other to allow EGY48 cells and RFY206 cells to hybridize at the overlap. 30°C overnight.

( 4 ) Replicate the YPD plates to YC Glc leu- his- trp - ura, YC Glc his- trp - ura- and YC Gal Raf leu- his- trp - ura- plates and conserve at 30°C for 3 d. All hybridized cells should grow on YC Glc his- trp - ura- plates. Growth on YC Gal Raf leu - his- trp - ura- plates is indicative of "bait" and "predator" interactions. Other plates were used as controls.

3.3.2 Yeast two-hybrid library screening

RFY206 containing the bait was hybridized with EGY48 containing the solo library and cells containing both solo and bait were selected. The application of yeast hybridization allows for efficient screening of multiple libraries [13] .

( 1 ) RFY206 residing pSH18-34 and LexA-bait plasmids were grown for 16 h at 30°C in 10 ml YC Glc his - ura- medium.

( 2 ) Cell concentration was determined by _OD600 using 1.0 _OD600 = 2.0 X ¹⁰⁷ cells/ml conversion. ¹⁰⁸ decoy cells were placed in microcentrifuge tubes and centrifuged at 10,000 r/min for 30 s on a microcentrifuge to allow the cells to rotate and settle, and the cells were allowed to suspend with 200 μl of medium.

( 3 ) Add ¹⁰⁷ lone-resident EGY48 cells to the decoy cells (see 3.1.7 ) and spread them out on a YP plate. Allow the cells to hybridize for 16 h.

( 4 ) Rinse the cells off the plates with 5 ml (1 ml at a time) of YPD medium. 100-fold dilution was used to measure the _OD600, and ¹⁰⁸ cells were spread on 5 YC Gal Raf leu- his- trp - ura- medium plates. These plates were conserved at 30°C for 3~4 d. The cells were washed from the plates.

( 5 ) Replicate colonies on YC Gal Raf leu- his- trp - ura- medium plates and YC Gal leu- his- trp - ura- medium plates. The true positive clones will only grow on YC Gal Raf leu- his- trp - ura- plates.

( 6 ) In a ventilated kitchen, open the plate lid and add enough chloroform to cover the surface of the plate. after 5 min pour the chloroform into a waste can and allow the remaining chloroform to evaporate for 10 min [ 20 ] .

( 7 ) Heat the agarose-phosphate solution (10 ml per dish) in a microwave oven to dissolve the agarose, and cool in a water bath to 50°C. Add 35 μl of 50 mg/ml X-gal solution and 10 ml of agarose-phosphate solution to a test tube, stir, and pour into the plate. conserve at 30°C. Observe the color. Observe the color. Pick out the clones showing blue color for further examination.

3.3.3 Specificity testing of isolated monomers

( 1 ) Cultivate the yeast cells of interest screened from the library in 1.5 ml YPD medium at 30°C for 16 h. Prepare DNA from yeast cells using Y- DER Yeast DNA Extraction Kit (Pierce).

( 2 ) Dialyzed yeast DNA is injected into E. coli KC8 cells by electroporation (see 3.1.4) and transformed cells are selected on LB-Ap plates.

( 3 ) Replicate colonies on basic trp-Km plates and conserve at 37°C. Select two colonies for inoculation. Two colonies were inoculated into 2 ml of LB-Ap medium and incubated at 37°C for 10 h. Plasmid DNA was extracted by standard methods. KC8 cells grown for more than 10 h often produce impure plasmids. The sequence of the unique fragments is determined by standard DNA sequencing (see Note 7).

( 4 ) Transform EGY48 with the B42-solitary plasmid and determine its interaction with various decoys by interaction hybridization (see 3.3.1).

3.3.4 Liquid-phase β-galactosidase test

Prior to biochemical measurements, the affinity of the lure for the monomers can be determined semi-quantitatively by the liquid-phase β-galactosidase test. This method is more quantitative than the plate test described in subsection 3.3.2. We have adapted this method to the 96-well plate format (see Note 8).

( 1 ) Transform the RFY206 Saccharomyces cerevisiae strain with pSH18-34, the LexA- decoy plasmid (a derivative of pEG202), and the B42 lone plasmid (a derivative of pYesTrp2). One colony was inoculated into 2 ml YC Glc his- ura-trp - and shaken at 30°C overnight.

( 2 ) Centrifuge slightly to remove the medium and suspend in warm YC Glc his-ura-trp- until the _OD600 value is 0.2. Shake at 30°C for 6 h. The _OD600 value is measured and loaded into microcentrifuge tubes at 350 μl each. Typically, we take three measurements at a time.

( 3 ) Mix 2 X β-galactosidase test solution with Y-Per (Pierce) 1 : 1 as working solution. Add 350 μl of working solution to each sample. Turn over several times to mix.

( 4 ) Conserve at 30°C while checking color. When yellow color appears, add 300 μl of 0.5 mol/L sodium carbonate to the centrifuge tube and mix thoroughly. Centrifuge the microcentrifuge tube at maximum speed for 1 min and determine the _OD420 value of the supernatant.

3.4 Cloning, expression, purification and biotinylation of the isolates

After screening the library for monomers with desired characteristics, the genes are transfected into an E. coli expression vector and the monomer is expressed and purified as an isolated protein. We typically obtained 10-15 mg of monomer per liter of culture. We also describe the procedure for biotinylating the monomer for ease of detection in immunochemical applications.

3.4.1 Cloning of the monomer

The lone biotin was cloned using the oligonucleotides NdeMetThrFNF (5'-CGGGATCCCATATATGCAGGTTTCTGATGTTCCGACCGACCTGGAAGTTGTTGCTG-3'; it contains mutations from Arg6 to Thr) and FNGKKGKR ( 5'-CCG ACTCGAGTTACTATTTACCTTTTTTACCGGTACGGTAGTTAATCGAG-3'), the solo genes of interest were amplified, then degraded with Nde I and Xho I and cloned into pET15b (Novagen) degraded with the same enzymes. Confirmation of the genes in the expression vector was done by sequencing.

3.4.2 Expression of the monomer

This procedure is suitable for 100 ml of culture medium, which should produce enough monomers for initial characterization.

( 1 ) Transform BL21 (DE3) cells (Novagen) with a lone expression vector (see Note 9).

( 2 ) Inoculate transformed cells into 10 ml of M9-Tryptone-Ap and conserve at 30°C for 10~16 h under strong shaking.

( 3 ) Inoculate 2 ml of pre-culture solution into 100 ml of pre-warmed M9-Tryptone-Ap and incubate at 37°C under strong shaking. When the _OD600 value reached 0.7, IPTG was added to a final concentration of 0.5 mmol/L. The incubator was incubated for 10~16 h at 30°C with strong shaking.

( 4 ) Conserve at 37°C for more than 3 h with strong shaking, and collect the cells by centrifugation. Cells can be stored at -20°C until use.

3.4.3 Purification of isolates

This procedure is suitable for 100 ml culture broth (see 3.4.2 ).

( 1 ) Suspend the cell precipitate in 6.4 ml of 50 mmol/L Tris-HCl at pH 8.0.

( 2 ) Add 64 μl of 50 mg/ml lysozyme and 128 μl of 50 mmol/L PMSF, mix and incubate at 37°C for 15 min. sonicate and suspend until no longer highly viscous.

( 3 ) Add 910 μl of 40 mol/L NaCl. Centrifuge at 27000 g (15000 r/min, SS34 rotor) for 10 min at 4°C. Collect the supernatant.

( 4 ) Place the protein solution in a Hi-Trap chelator column (1 ml; Amersham-Pharmacia Biotech) loaded with 0.1 mol/L nickel chloride and equilibrated with Buffer B. The protein solution is then separated from the protein solution by a 6 ml buffer. The column is washed with 6 ml of Buffer B followed by 6 ml of Buffer B containing 60 mmol/L imidazole.

( 5 ) Elute the monomer with 6 ml of buffer C. Collect 0.5~1 ml of buffer B and elute with 6 ml of buffer C. Collect 0.5-1 ml of each eluate into a microcentrifuge tube. Analyze the eluate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

3.4.4 Biotinylation of the monomers

We attached biotin to the amino group of Lys. This is not site-specific, but Lys, which clusters at the C-terminal extension, should be the most susceptible site for modification. This method can be used to attach other fragments such as fluorescent dyes.

( 1 ) Complete steps (1 )~( 4 ) of the purification operation in subsection 3.4 3.

( 2 ) Wash the column with 10 ml of Buffer D.

( 3 ) Dissolve 1 mg of D-biotin- ε - aminocaproic acid light- sulfosuccinimide lipid (BoehringerMannheim) in 50 μl of Dimethyl Sulfoxide and dilute to 3 ml of Buffer D. 1 ml of the solution was injected into the column and held at room temperature in the dark for 1 h. The reaction was repeated twice.

( 4 ) Wash the column with 6 ml of Buffer B and elute the monomer as described in step (5) of section 3.4.3.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/