A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex

Abstract

A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

Figure 1

The structure (A) and sequence (B) of TAP tag (CHH) consisting of calmodulin binding peptide (CBP), six histidine residues (HIS6), and three copies of the hemagglutinin epitope (HA3), flanked by NotI sites.

Figure 2

TAP of Clb2–Cdc28 complex. Untagged Clb2 (odd-numbered lanes) and Clb2-CHH (even-numbered lanes) were expressed under the control of the GAL promoter in strains YSH16 and YSH19. (A) Untagged (lane 1) or tagged (lane 2) cell extracts were purified on a nickel (Ni) affinity resin, and visualized after 10% SDS–PAGE and silver staining. (B) Untagged (lane 1) or tagged (lane 2) cell extracts were purified on a calmodulin (Cm) affinity resin. Pooled eluted fractions from the calmodulin affinity resin were further purified by immunoprecipitation (IP) with 12CA5 monoclonal antibody on protein A beads (lane 3, untagged control; lane 4, Clb2-CHH). Proteins were separated using 10% SDS–PAGE and visualized by silver staining. Most of the material visible in lane 3 is heavy (H) or light (L) chain IgG.

Figure 3

Western analysis of proteins associated with Clb2–Cdc28 complex. Cell extracts prepared from untagged Clb2 (lane 1) and Clb2-CHH (lane 2) were subjected to sequential purifications on calmodulin affinity resin followed by immunoprecipitation with 12CA5 monoclonal antibody on protein A beads. Immunoprecipitates were applied to 10% SDS–PAGE gels and immunoblotted with antibodies against HA (monoclonal 12CA5), Sic1 (rabbit polyclonal) and Cdc28 (rabbit polyclonal).

Figure 4

Kinase activity of various fractions. Untagged (fractions 1–3) or CHH-tagged Clb2 (fractions 4–9) was purified by binding to calmodulin or nickel resin, eluted, then immunoprecipitated with 12CA5. Equal proportions of various fractions were assayed for protein kinase activity using [γ-³²P]ATP, peptide substrates and a filter binding assay (Materials and Methods). Fraction 1, untagged Clb2, flow-through from calmodulin resin. Fraction 2, untagged Clb2, eluate from calmodulin resin. Fraction 3, untagged Clb2, immunoprecipitate (IP) of eluate. Fraction 4, Clb2-CHH, flow-through from calmodulin resin. Fraction 5, Clb2-CHH, eluate from calmodulin resin. Fraction 6, Clb2-CHH, IP of eluate from calmodulin resin. Fraction 7, Clb2-CHH, flow-through from nickel resin. Fraction 8, Clb2-CHH, eluate from nickel resin. Fraction 9, IP of eluate from nickel resin. The data are means of triplicate determinations. Standard deviation in each case was <15% of the mean.

Figure 5

Resolution of TAP Clb2–Cdc28 complex on silver-stained gel. Extracts from 12 l cultures of yeast strains YSH16, YSH19 and YSH24 carrying untagged Clb2 and Cdc28 (lane 1), Clb2-CHH and Cdc28 (lane 2), or Clb2-CHH, Cdc28 and Cdc28K40R (lane 3), respectively, were purified on calmodulin affinity resin and then immunoprecipitated with 12CA5 monoclonal antibody. Purified proteins were resolved using 10% SDS–PAGE and proteins were visualized by silver staining. Silver-stained protein band nos 1–23 (lane 3) were excised, digested with trypsin and fragments were used for the identification of proteins by mass spectrometry.