Site-Specific Fluorescent Labeling of Tubulin - Methods in Cell Biology - page 6

1.2.1.2 Tubulin tyrosine ligase
Transform competent Escherichia coli BL21 with expression vector EX-T8583-B05
(pReceiver 05 containing GST-TTL gene and ampicillin-resistant gene) and grow

gmL 1 Ampicillin containing LB (LB-Amp) agar. Inoculate trans-
formed cells in LB-Amp broth and allow them to grow overnight at 37 C. Mix
the overnight culture with fresh medium (1:10) and incubate in a shaker incubator
at 37 C. At OD 600 ¼

them on 100 m

0.4-0.5, induce protein expression with 1 mM IPTG. Return
to the shaker incubator for 20 h and then centrifuge the broth at 3000 rpm for
20 min at 4 C. At this point, the pellet may be stored at 50 C for many months.

Resuspend the cell pellet by sonication in TE buffer containing 0.3 mg of PMSF
per mL. (We use 20-30 mL buffer for cell pellet obtained from a 400 mL culture.)
Perform six cycles of sonication. Each cycle should include 10 s of sonication
followed by 15 s incubation on ice. Centrifuge the resultant lysate at 3000 rpm
for 20 min to remove the debris. TTL-GST can then be isolated by affinity
chromatography.
Wash glutathione Sepharose 4B with PBS following the manufacturer's instruc-
tions. Mix the cleared lysate with glutathione Sepharose 4B ( L for the

400 mL culture volume) and incubate on a rocker at 4 C for 45 min. Remove un-
bound protein by batch-washing in the following manner. Pellet the resin by
centrifuging at 1300 rpm or 300

300 m

g for 2 min. Add approximately 3 mL of PBS
per mL of resin volume, mix by gentle inversion three times, and centrifuge the sus-
pension at 1300 rpm or 300 g for 2 min. Repeat this step four times. Resuspend the

resin in PBS (approximately 2 mL PBS per mL of resin) and pour the mixture into a
5-mL column. Elute the bound protein with five resin volumes of elution buffer.
Collect elution fractions equivalent to the resin volume and check each fraction
for protein. Pool the fractions containing purified protein and determine the protein
concentration. Store the protein as aliquots at 50 C until use.

The GST portion of the expressed protein can be removed by TEV protease
(Sigma). We use the entire fusion protein (GST-TTL) for further experiments.

1.2.2 Tubulin detyrosination
Detyrosinate tubulin by treating purified tubulin in PME buffer with CPA ( final con-
centrations: 0.1mg per mL CPA, 3mg per mL tubulin) for 10 min at 37 C. Submit
the mixture to rapid gel filtration with Sephadex G-75 in TTL buffer. We use 1mL
syringe spin columns following the method of Penefsky (1979) . This process also
removes the cleaved tyrosine and CPA.

1.2.3 Coupling tyrosine derivative to the C-terminus of -tubulin

a

1.2.3.1 Ligand preparation
1.2.3.1.1 L -Tyrosine
Make 100 mM tyrosine stock solution by dissolving appropriate mass of tyrosine in
250 mM NaOH solution.