2.2 Methods
2.2.5 Introduction of a C-terminal TAP tag to the yeast protein Cbp20 by homol-
The applied protocol described in the sections below follows established procedures[92] with slight modifications unless noted otherwise. Dr. Kum-Loong Boon (Department of Cellular Biochemistry) gave technical support in the experiments.
2.2.5.1 Generation of DNA
DNA template was the pBS1539 plasmid which was constructed to introduce a C-terminal TAP tag and contains a URA3 selective marker from Kluyveromyces lactis [93]. The pBS1539-psc plasmid used here, which contains a PreScission instead of the TEV cleavage site, was provided by Dr.
Kum-Loong Boon.
The TAP cassette was amplified by polymerase chain reaction (PCR). Primers, reaction mix and PCR program are listed below. In the primer sequences, regions homologous to the pBS1539-psc plasmid are underlined. The 5’ ends are homologous to the target geneCBP20.
forward primer:
5’-TCA GAC CAG GTT TCG ATG AAG AAA GAG AAG ATG ATA ACT ACG TAC CTC AGT CCA TGG AAA AGA-GAA GAT-3’
reverse primer:
5’-TAT ATA TAT ATC TGT GTG TAG AAT CTT TCT CAG ATA TAA ATT-GAT TGA TTT ACG ACT CAC TAT AGG GCG A-3’
Formation of PCR product was confirmed by agarose gel electrophoresis. The PCR product was isolated with phenol-chloroform extraction. To this end, 240μl PCI solution (1:1 V:V) were added and mixed by vortexing. Phases were separated by centrifugation at 13 000 rpm for 10 min. DNA was precipitated from the aqueous phase with 2.5 volumes of ethanol and 1/10 volume of 3 M NaOAc. The DNA was pelleted by centrifugation, air dried, and subsequently dissolved in 35μl H2O.
2.2 Methods 39 2.2.5.2 Transformation
To construct the yeast strain expressing TAP tagged Cbp20 (Cbc2p), the PCR product containing the C-terminal TAP tag cassette was transformed into yeast strain BJ2168 with the lithium acetate (LiOAc) method[94, 95].
Transformation mix was prepared by mixing 35μl DNA solution, 36μl 1 M LiOAc and 240μl PEG3350 solution (50% w/V), and 40μl denatured fish sperm carrier DNA (2 mg/ml; DNA, MB-grade from fish sperm, Roche, Mannheim, Germany).
Competent yeast cells for transformation were prepared from a 50 ml overnight culture grown to an OD600 of 0.6-1.0. Cells were spun down by centrifugation at 4 000 rpm and 4◦C for 3 min, subsequently washed with 1 ml H2O and centrifuged as above. Cells were resuspended in 400μl 100 mM LiOAc.
50μl cell suspension were incubated with the transformation mix on a rotating wheel at room temperature for 30 min, followed by a heat shock at 42◦C for 20 min. Cells were pelleted by brief centrifugation, the transformation mix was removed and cells were resuspended in 125μl H2O. Cells were plated on –URA selective plates and incubated for 2-3 days at 30◦C. The transformants were restreaked onto a fresh –URA selective plate for further validation.
2.2.5.3 Yeast colony PCR
Correct insertion of the TAP tag construct into the yeast strain was confirmed by yeast colony PCR. Forward primer was the same as above, homologous to the chromosomal sequence and the inserted TAP cassette (latter underlined in primer sequence). The reverse primer was homologous to the ProtA sequence (underlined). PCR products were verified by agarose gel electrophoresis.
forward primer:
5’-TCA GAC CAG GTT TCG ATG AAG AAA GAG AAG ATG ATA ACT ACG TAC CTC AGT CCA TGG AAA AGA-GAA GAT-3’
reverse primer:
5’-CCT TAA ATC AGG TTG ACT TCC CCG CGC A-3’
PCR mix (14 samples)
PCR program
2.2.5.4 Confirmation of TAP tag inclusion by Western blot
Yeast clones confirmed by yeast colony PCR were further investigated by Western blotting. For sample preparation, 2 ml overnight cultures cultivated in YPD broth were harvested by centrifu-gation at 3 500 rpm and 4◦C for 4 min. Cell pellets were resuspended in 500μl 0.2 M NaOH and incubated on ice for 10 min. Subsequently, 27.5μl TCA (100% w/V) were added and the cell lysate was further incubated on ice for 10 min. Proteins were spun down at 13 000 rpm for 30 s. Protein pellets were resuspended in 35μl dissociation buffer (0.1 M Tris pH 6.8, 4 mM EDTA pH 8.0, 4%
SDS, 20% glycerol, 20 mM DTT). After addition of 15μl 1 M Tris, the sample was boiled at 95◦C for 10 min. Cell debris were removed by centrifugation (10 s at 13 000 rpm). Proteins were sepa-rated by SDS-PAGE, and the TAP tagged protein was detected by Western blotting by peroxidase anti-peroxidase antibody and visualized by enhanced chemiluminescence (ECL).
2.2.5.5 Confirmation of TAP tag inclusion by sequencing
The yeast strain confirmed to express TAP tagged protein by Western blotting was further verified by DNA sequencing. Yeast DNA for PCR prior to sequencing was prepared from 50 ml yeast culture cultivated in YPD. Cells were harvested at 4 000 rpm and 4◦C for 3 min. Cell pellets were washed once with 10 ml deionized water, centrifuged as above and resuspended in 10 ml SE buffer (0.9 M sorbitol, 0.1 M EDTA pH 8.0). 50μl lyticase (20 mg/ml) were added and the cell suspension was incubated for 30–60 min at room temperature. Cells were spun down at 5 000 rpm for 5 min, resuspended in 500μl lysis buffer (0.1 M Tris pH 8.0, 50 mM EDTA, 1% SDS) and 32μl 4 M NaCl were added. In order to break the cells, glass beads were added to the cell suspension and the sample was vortexed for 1 min. Cell debris and glass beads were removed by centrifugation at 5 000 rpm for 5 min. DNA was isolated by PCI extraction and ethanol precipitation.
Two PCRs were prepared as described in 2.2.5.1 with the same forward primer. In the first PCR, the same reverse primer as in 2.2.5.3 was used. The reverse primer for the second PCR is listed below.
It is homologous to the URA3 sequence in the TAP cassette. PCR products were visualized by agarose gel electrophoresis and sequenced (SEQLAB Sequence Laboratories, Göttingen, Germany).
The obtained sequencing results showed no mutations in the coding region.
reverse primer 2:
5’-AGA GAA TCA GCG CTC CCC AT-3’
2.2 Methods 41