EXPRESSION OF PLANT RECEPTOR-LIKE KINASE LEUCINE RICH REPEAT REGIONS IN E. COLI
Andrea Corns, Emily Heffernen, Ara May Hubbard, Paula Macksey, Kelsey Pigg, Andrew Platt, Maria Stieff,
Julia
Tucci, Guru Rao, and Heidi Sleister. Biology and BCMB, Drake University; BBMB, Iowa State University
ABSTRACT
EXPERIMENTAL METHODS
Plants respond to external stimuli through signal transduction pathways.
An external signal binds the extracellular domain of a transmembrane
receptor-like kinase (RLK) protein, and the signal is transmitted inside
the cell through the RLK’s kinase domain. Two important plant RLKs
are Arabidopsis CLV1 and rice Xa21. CLV1 regulates plant meristem
growth and development, and Xa21 provides resistance to the
pathogenic bacterium Xanthomonas oryzae. These functions are
initiated by a peptide ligand binding a leucine-rich repeat (LRR) region
in the extracellular domains of CLV1 and Xa21. Peptide phage display
technology will be used to isolate additional peptide ligands that bind
CLV1 and Xa21 LRRs. To this end, the DNAs encoding these LRRs are
being cloned into vector pSUMO_Kan for soluble expression in E. coli
as a fusion with SUMO protein. Fusion proteins will be isolated from E.
coli cells by immobilized metal affinity chromatography.
SIGNIFICANCE: If successful, this project will be the first reported
example of soluble expression of a large plant LRR region in E.
coli. In the future, soluble LRR protein will be used as the target
in peptide phage display to isolate peptides that bind the LRR.
RECEPTOR-LIKE KINASES CLV1 AND Xa21
Panning Phage Display Library for Peptides
that Bind CLV1 / Xa21 LRR Regions
PCR amplify CLV1 and Xa21 LRR
regions to incorporate protein tags
(Halo or FLAG) and 5’ and 3’ EcoRV
restriction sites
Cut PCR product with EcoRV,
gel purify
A library of phage, each
displaying a different
peptide, is allowed to bind
LRR-HaloTag fusion protein
immobilized on HaloLink
magnetic beads.
phage
CLV1 / Xa21 LRR region
Linearize vector pSUMO_Kan with BsaI,
polish ends with Pfu DNA Polymerase,
dephosphorylate, gel purify
peptide
LRR
HaloTag
magnetic bead
Ligate prepared vector + prepared CLV1/Xa21 LRR
Screen transformants by colony PCR, restriction digests, and sequence analysis
Express LRR region fused with SUMO in bacteria
Isolate soluble SUMO-LRR fusion protein by immobilized metal affinity chromatography (IMAC)
Figure 2. Methods to clone and express CLV1 and Xa21 LRRs as fusions with SUMO protein.
PCR Primer Name
SUMO-CLV1-F2
Primer Length
(Nucleotides)
Primer Sequence
AGGTGATATCGACGACGATGCTCGTGTTATC
31
SUMO-CLV1-Halo-R2 CTAGGATATCTTAACCGGAAATCTCCAGAG
30
SUMO-CLV1-F2
31
AGGTGATATCGACGACGATGCTCGTGTTATC
SUMO-CLV1-Flag-R2 CTAGGATATCTTACTTATCGTCGTCATCCTTGTAATCGGAAGTTTCGTTGAAC
53
SUMO-Xa21-F2
27
AGGTGATATCCGCCGGCACCCACACAG
SUMO-Xa21-Halo-R2 CTAGGATATCTTAACCGGAAATCTCCAGAGTAG
33
SUMO-Xa21-F2
27
AGGTGATATCCGCCGGCACCCACACAG
SUMO-Xa21-Flag-R2 CTAGGATATCTTACTTATCGTCGTCATCCTTGTAATCTCGAGGCAGATGTAGATC
55
PCR Conditions
PCR Product
Size (Basepairs)
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 50oC, 3
min 72oC]; 10 min 72oC
2582 bp
2 min 94oC; 35 cycles [45
o
o
sec 94 C, 45 sec 50 C, 3
min 72oC]; 10 min 72oC
1664 bp
2 min 94oC; 35 cycles [45
o
o
sec 94 C, 45 sec 52 C, 3
min 72oC]; 10 min 72oC
2663 bp
2 min 94oC; 35 cycles [45
sec 94oC, 45 sec 52oC, 3
min 72oC]; 10 min 72oC
1745 bp
Panning process is repeated ~3 times.
Transform ligation mixture into E. coli competent cells
PROJECT AIM & SIGNIFICANCE
PROJECT AIM: To express and purify the leucine rich repeats
(LRRs) from plant receptor-like kinases (RLKs) CLV1 and Xa21 as
soluble fusions with SUMO protein in E. coli.
FUTURE WORK
Unbound phage are
washed away.
Specifically-bound phage
are eluted, amplified, and
used for the next round of
panning.
After ~4 rounds of panning,
individual clones are
isolated and sequenced.
Table 1. PCR primers and conditions
N-terminal region
Flanking paired Cys
SUMMARY
CLV1 is important for shoot apical meristem
development. CLV’s natural ligand is a 13amino acid arabinosylated peptide derived
from 96-amino acid CLV3
(RTVPSGPDPLHHH).
• Construction of CLV1 and Xa21 LRRs as fusions with SUMO is in
progress. The vector and LRR inserts were successfully isolated and
ligated. Transformants are being screened for presence of the insert.
RESULTS
LRR region
Flanking paired Cys
Transmembrane domain
Kinase domain
N-terminal region
VISLNVSFTPLFGTISPEIGMLTHLVNL
NISNNGNLTGTFPGEILKAMVDLEVL
SFGGNFFSGEIPESYGDIQSLEYL
MYIGYYNSYTGGVPPEFGGLTKLEIL
FLHINNLTGHIPPELSGLVSLKSL
NLFRNNLYGQIPEAIGELPKLEVF
DVSDNHLTGLIPKDLCRGEKLEML
RIVKNLLNGTVPAGLFNLPLVTII
YLSNNWFSGEIPPAIGNFPNLQTL
NTSANNITGGIPDSISRCSTLISV
NISGNQLTGSIPTGIGNMTSLTTL
TLAANNFTGELPLEMKSLTSLKVL
DTYNNNFNGKLPPEMSELKKLKYL
GLNGAGLSGKSPAFLSRLKNLRE
DMASCTLTGEIPTSLSNLKHLHTL
DLSINQLTGEIPQSFINLGNITLI
EVWENNFTLQLPANLGRNGNLIKL
ILSNNFFFGPIPEELGKCKSLTKI
ELTDNFFSGELPVTMSGDVLDQI
FLDRNRFRGNIPREIFELKHLSRI
DLSRNRINGEIPKGINNVKNLGTL
DLSFNDLSGRVPLGGQFL
Xa21 is important for resistance to
Xanthomonas oryzae which causes rice
bacterial blight disease. A 17-amino acid
sulfated peptide derived from 194-amino acid
Ax21 (AENLSYNFVEGDYVRTP) functions as a
ligand for Xa21.
LRR region
Transmembrane domain
Kinase domain
VVKLLLRSSNLSGIISPS
LSRLSRLQLLELSDNSIQGSIPAA
GASLKHLSNLYLYKNGLSGEIPSA
LGQLSSLLTMNLGQNNLSGMIPNS
FKTLHLLEVIDMGTNRFHGKIPAS
FGRLRNLTELYLWRNLFQTREQDDWGFISD
SNLSTSLSFLALELNKITGSIPKD
LGRLKNLGILLAYENNLSGSIPLA
LSNLTNLLSLGLSTNNLSGPIPSE
IGHLKNLVEFHAESNRLSGKIPNT
LGQLKGLETLDLSSNNLSGQIPTS
IGAFAAASG ISIQGNAKLCGGIP
LGNLSFLRELDLGDNYLSGEIPPE
IGACTKLTSLDLSHNQLRGMIPREI
LGNLTSLQEFDLSFNRLSGAIPSS
IWNLSSLRAFSVRENKLGGMIPTNA
VANASHLTVIQIYGNLFSGIITSG
LTNCSKLQTLNLGENNLGGVLPNSF
IGNLIGLQHLYLCNNNFRGSLPSS
IGNLTELNILLLGTNKFSGWIPYT
LFNIQTLSIMINVSKNNLEGSIPQE
LGDCQLLRYLYLQNNLLSGSIPSA
LADITMLHSLNLSFNSFVGEVPT
C-terminal domain
Figure 1. Structures of CLV1 and Xa21 proteins. The leucine rich repeat (LRR) region is
the extracellular portion of the protein that binds a ligand to initiate signal transduction.
1
1
2
3
4
5
6
7
2
3
8
1. pSUMO vector cut with BsaI
2. Uncut pSUMO vector
3. Lambda-HindIII marker
• Validation of a phage display combinatorial approach to isolate
peptide ligands for plant RLKs CLV1 and Xa21 may lead to general
utility of this system for signal transduction in plants and animals.
2.3 kb
2.0 kb
Figure 3. 0.8% agarose gel of CLV1(LRR) and
Xa21(LRR) PCR products. The size of each
PCR product (lanes 2-5, marked with arrows)
is correct.
1.
2.
3.
4.
5.
6.
7.
8.
Lambda-HindIII marker
Xa21(LRR)-Halo PCR product
Xa21(LRR)-FLAG PCR product
CLV1(LRR)-Halo PCR product
CLV1(LRR)-FLAG PCR product
Uncut plasmid template for CLV1 PCR reaction
Uncut plasmid template for Xa21 PCR reaction
Lambda-HindIII marker
Figure 4. Preparation of pSUMO vector. 0.8%
agarose gel of BsaI-cut and uncut pSUMO.
CLV1(LRR)
CLV1(LRR)
Xa21(LRR)
Xa21(LRR)
Halo
FLAG
http://www.google.com/imgres?imgurl=http://www.carto.net/neumann/travelling/japan_2004_09/04_kyoto_2004_09_12/29_rice_p
lant.jpg&imgrefurl=http://www.carto.net/neumann/travelling/japan_2004_09/04_kyoto_2004_09_12/&h=614&w=819&sz=65&tbnid
=6awJqYynLZFAMM:&tbnh=108&tbnw=144&prev=/search%3Fq%3Drice%2Bplant%26tbm%3Disch%26tbo%3Du&zoom=1&q=ri
ce+plant&usg=__of-NUAjqtzK9TJpvVmYzMzpNd_M=&sa=X&ei=azibTbOnCoz2swO94ImbBA&ved=0CDEQ9QEwAA
http://faculty.washington.edu/ktorii/ERECTA04.html
ACKNOWLEDGEMENTS & REFERENCES
We thank Pamela Ronald (Univ. California, Davis) for Xa21-containing plasmid pCR504 and the Meyerowitz
laboratory (California Institute of Technology) for CLV1-containing plasmid pKR123. This work was funded
by Iowa-NASA EPSCoR, Drake University, and Iowa State University.
Halo
FLAG
Figure 5. Protein expression constructs.
1 Morris, E. and J. Walker. 2003. Receptor-like protein kinases: the keys to response. Curr Opin Plant Biol. 6:339-42.
2 Walker, J. 1994. Structure and function of the receptor-like protein kinases of higher plants. Plant Mol Biol. 26: 1599-609.
3 Ito, Y. et al. 2006. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313: 842-5.
4 Kondo, T. et al. 2008. Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides. Biochem Biophys Res
Commun. 377:312-6.
5 Trotochaud, A. et al. 2000. CLAVATA3, a Multimeric Ligand for the CLAVATA1 Receptor-Kinase. Science 289: 613-7.
6 Lee, et al. 2009. A Type I–Secreted, Sulfated PeptideTriggers XA21-Mediated Innate Immunity. Science 326: 850-853.