Proteomics

1. Presented by:
Mesele Tilahun
(Integ.2nd year)
Advisor: MoonJae Sun
May 18, 2021
KRIBB,UST, S.Korea

2. Contents
1. INTRODUCTION: GRAS Protein
2. GRAS Protein production
3. GRAS Protein sequence analysis

3. Introduction
• Proteins – large molecules that are required for the structure,
function, and regulation of living cells
• Are complex molecules built of chains of amino acids
• Have electrical charge that causes them to interact with other atoms and
molecules
• Hydrophilic – water loving
• Hydrophobic – water hating
• Protein Sequencing
• determine the primary structure, the sequence of amino acids
• X-ray Crystallography
• Used to determine the complex tertiary and quaternary structures

4. GRAS proteins
 GRAS proteins- plant-specific protein family
Named first 3 members
Gibberellic Acid Insensitive (GAI)
 Repressor of GAI
Scarecrow (SCR)
 Found in more >294 embryophyta
 play key roles in the transcriptional regulation and signaling
transduction related to plant growth and development
 divided into >10 subfamilies, of which SCL4/7 is important
subgroup and functions in response to environmental stresses.

5. GRAS proteins Structure
GRAS proteins include
a variable N-terminal domain- constitute unfoldome (disordered regions)
facilitate molecular recognition with different interacting partners &
contribute to divergent biological functions.
highly conserved C-terminal domain/ GRAS domain family, these proteins
share a similar function and common mode-of-action.
-Protein in this family minimally have CDD
 The structure contains 5 highly conserved motifs.
• The two LRI and LRII directly flank the VHIID motif, PFYRE and SAW
• LR motifs and VHIID motif could be important for protein or DNA
interactions in most members of the GRAS.

6. Overall Structure, Sequence, and Secondary Structure of GRAS Domain of Os-SCL7

7. Protein Production
• Production of Os-SCL7 involves
• Upstream processing- the actual expression of the protein in the cell
• Downstream processing- purification of the protein and verification
 Many members of GRAS proteins studied, the functional mechanism of
GRAS proteins is still unclear.
 Structural descriptions of GRAS proteins may deeply clarify the functional
mechanism of this family.
 However as yet little structural analyses have been reported, mainly due to the
difficulties in obtaining sufficient quality and quantity of GRAS proteins.

8. Expression and purification of the GRAS domain of Os-SCL7 from rice
1. Clone gene encoding GRAS domain Os-SCL7 from rice  pET32a
2. Rec. plasmid E.coli strain BL21 by heat shock
3. Pick a single colony  LB medium posses antibiotic
4. Induce protein expression with IPTG overnight
5. Harvest the cells by cfg  sonication (placing on ice)  cfg
6. Transfer and add imidazole into the supernatant (reduce non-specific proteins
binding)
7. Load the sample  nickel-sepharose affinity resin
8. Elute the bound protein with lysis buffers
9. Eluted protein mixed with loading buffer to SDS-PAGE
OsSCL7 GRAS domain protein fused with TRX and 6His-tag

9. Expression and purification…
10. Add TEV protease to the eluted proteins of interest to remove the TRX
& 6His-tag from OsSCL7 GRAS domain.
Note: In this step, the lysis buffer is used to dialyze imidazole to a low con. (< 5 mM)
11. Load the cleaved protein  nickel-sepharose affinity resin again. Target
proteins are flow through and subsequent 20 mM imidazole eluent.
12.Then load the sample HiLoad 16/600 Superdex200 pre-equilibrated
with chromatography buffer.
13. Pool the fractions containing target protein & measured by Bradford
method on spectrophotometer device

10. Data analysis
Lane 1: The aggregates
Lane 2: The flow
Lane 3: Eluted with 20 mM imidazole;
Lane 4: Eluted with 500 mM imidazole;
M: Marker. The arrow indicates the similar MW with
the predicted MW (59 kDa) of OsCL7 GRAS domain
(41.5 kDa) + Trx-tag (17.5 kDa);
Lane1: The flow of the second nickel-sepharose affinity
resin purification (TRX and 6His-tag cut off from the
OsSCL7 GRAS domain), according to the MW of
OsSCL7 GRAS domain, around 42 kDa;
Lane 2: Elution with 20 mM imidazole, which also
contains the OsSCL7 GRAS domain;
M: Marker.
15% SDS-PAGE analysis of purified OsSCL7 GRAS domain by nickel-sepharose affinity resin stained with Coomassie Brilliant Blue.

11. Purification of OsSCL7 GRAS domain by size-exclusion chromatography.
a. Size-exclusion chromatography (HiLoad 16/600 Superdex 200 column), the dimer and monomer of OsSCL7 GRAS domain appear at 72.26 and 79.68 ml respectively, their
corresponding molecular weights are 84 kDa and 42 kDa.
b. 15% SDS-PAGE analysis of purified OsSCL7 GRAS domain from fractions of the observed peak in size-exclusion chromatography, showing high purity.

12. Sequence analysis
NCBI Reference Sequence: NM_001057650.1
Oryza sativa
MAYMCADSGNLMAIAQQVIQQQQQQQQQQQRHHHHHHLPPPPPPQSMAPHHHQQKHHHHHQQMPAMPQAPPSSHGQI
PGQLAYGGGAAWPAGEHFFADAFGASAGDAVFSDLAAAADFDSDGWMESLIGDAPFQDSDLERLIFTTPPPPVPSPP
PTHAAATATATAATAAPRPEAAPALLPQPAAATPVACSSPSPSSAD
201
ASCSAPILQSLLSCSRAAATDPGLAAAELASVRAAATDAGDPSERLAFYFADALSRRLACGTGAPPSAEPDARFASD
ELTLCYKTLNDACPYSKFAHLTANQAILEATGAATKIHIVDFGIVQGIQWAALLQALATRPEGKPTRIRITGVPSPL
LGPQPAASLAATNTRLRDFAKLLGVDFEFVPLLRPVHELNKSDFLVEPDEAVAVNFMLQLYHLLGDSDELVRRVLRL
AKSLSPAVVTLGEYEVSLNRAGFVDRFANALSYYRSLFESLDVAMTRDSPERVRVERWMFGERIQRAVGPEEGADRT
ERMAGSSEWQTLMEWCGFEPVPLSNYARSQADLLLWNYDSKYKYSLVELPPAFLSLAWEKRPLLTVSAWR
578
Proto param= https://web.expasy.org/protparam/
https://web.expasy.org/peptide_cutter/
predicts potential cleavage sites cleaved by proteases
No. of
cleavage
Cleavage site
Arg-C
proteinase
29 16 33 45 56 57 73
Proteinase K 212 1 5 7 8 11 12 17 18
Number of amino acids: 378
Molecular weight: 41514.36
Theoretical pI: 5.47
https://string-db.org/ PDB code = Os-SCL7 =5hyz
https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi