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Eluted calmodulin beads

FIGURE 36.1. An overview of the steps in TAP.

FIGURE 36.1. An overview of the steps in TAP.

sequence in TAP-tag, and the cleavage occurs between Q and G amino acid residues (Figure 36.2).

The N-terminal and C-terminal TAP-tags have reverse orders of the domains in the TAP-tag to allow for the TEV cleavage of only the ProA domains. These synthetic TAP-tags also include the castor bean catalase intronl, which is known to increase gene expression in rice plants [20]. The TAP-tag cassettes were cloned into a binary vector containing gateway recombination sites (Invitrogen) either N-terminal to the attRl sites or C-terminal to the attR2 site. The N-terminal TAP-tag is designed to be in reading frame 1 of the downstream attBl site, and thus in this case the AUG start codon of the target gene is separated from the 3' end of CBP domain by a 19-amino-acid structure consisting of exons flanking the catalase intron, junction regions, and attBl site. The C-TAP tag is also designed to be in reading frame l upstream of the attB2 site, and so in this case a 20-amino-acid structure consisting of attB2, junction region, and catalase exons separates the 3' end of the target gene and the 5' end of the CBP domain (Figure 36.3). These l9- or 20-amino-acid linkers serves as hydrophilic spacers between the TAP-tag and the target protein, presumably increasing the steric availability of the CBP region for binding to CaM beads. The N-TAP and C-TAP tag¬ęGateway cassettes were cloned in the pPTN289 (Tom Clemente, unpublished data) binary vector. The N-terminal or C-terminal TAP-tag fusion cassettes expressed from ECaMV 35S promoter are available as HindIII restriction fragments for transfer to other vectors. In our lab this TAP-tag fused with different rice protein kinases has been expressed in rice using the maize Ub promoter.

N-TAP tag:

atg gtg gtc gac aac aag ttc aac aag gaa cag caa aac gcg ttc tac gag ate ctg cac ctg ccg aac ctc aac gaa gag caa cgc aat gcc ttc Pro A:mvVDNKFNKEQQNAFYEILHLPNLNEEQRNAF ate cag age ctg aag gac gat ccg tcc cag age gcc aac ctg ctc gcc gaa gcg aag aag ctg aac gac gcg cag gca ccg aag IQSLKDDPSQSANLLAEAKKLNDAQAPK

atg cat gtg gac aac aag ttc aac aag gag caa cag aac gcc ttc tac gag ate ctc cat ctc cca aac ctg aac gag gaa cag cgt aac gcg ttc Pro A:mhVDNKFNKEQQNAFYEILHLPNLNEEQRNAF ate cag tcc ctc aag gat gac cca age caa tec gcg aac ctc ctg gcg gag gcc aag aag etc aac gat gcc caa gcc cca aag IQSLKDDPSQSANLLAEAKKLNDAQAPK

gac tac gac ate cca acc act gcc age gag aac ctg tac ttc cag ggc gag ctg aag acc gca get ctg gca cag cac gac gac gtg TEV: DYDIPTTASENLYFQGELKTAALAQHDDV

ggc age act agt atg gag age age aga tgg aag age aac ttc ate gca gtg age gcc gca aac ege ttc aag aag ate age tcc age ggc gca ctg CBP: gstsMESSRWKSNFIAVSAANRFKKISSSGAF

C-TAP tag:

atg gag age age aga tgg aag age aac ttc atc gca gtg √Ęge gcc gca aac cgc ttc aag aag atc age tcc age ggc gca ctg CBP: MESSRWKSNFIAVSAANRFKKISSSGAL

gac tac gac atc cca acc act gcc age gag aac ctg tac ttc cag ggc gag ctg aag acc gca gct ctg gca cag cac gac gag gtg TEV: DYDIPTTASENLYFQGELKTAALAQHDEV

gtc gac aac aag ttc aac aag gaa cag caa aac gcg ttc tac gag atc ctg cac ctg ccg aac ctc aac gaa gag caa cgc aat gcc ttc Pro A:VDNKFNKEQQNAFYEILHLPNLNEEQRNAF atc cag age ctg aag gac gat ccg tcc cag age gcc aac ctg ctc gcc gaa gcg aag aag ctg aac gac gcg cag gca ccg aag IQSLKDDPSQSANLLAEAKKLNDAQAPK

Pro A: atg cat gtg gac aac aag ttc aac aag gag caa cag aac gcc ttc tac gag atc ctc cat ctc cca aac ctg aac gag gaa cag cgt aac gcg ttc mhVDNKFNKEQQNAFYEILHLPNLNEEQRNAF atc cag tcc ctc aag gat gac cca age caa tcc gcg aac ctc ctg gcg gag gcc aag aag ctc aac gat gcc caa gcc cca aag tga IQSLKDDPSQSANLLAEAKKLNDAQAPK*

FIGURE 36.2. Structure of N-terminal and C-terminal TAP-tags.

L Br

E35SPro J NTAP TEVL

Target gene |35ST | Bar NosT attB1 attB2 Nos Pro

Catalase intron

C-TAP tag R Br

L Br

E35SPro Target gene attB1 attB2

CTAP 35ST | Bar NosT Nos Pro

Catalase intron

FIGURE 36.3. Structure of the T-DNA expressing TAP-tagged target protein. The ECaMV 35S promoter (E35SPro) and CaMV 35S polyadenylation region (35ST) are used to express the TAP-tagged gene. TEVL in N-TAP tag figure designates the TEV untranslated leader. R Br and L Br represent right and left borders, respectively. See insert for color representation of this figure.

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