Our Goals

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Our Goals
Our Goals

• Optimize native solubility of our proteins Obtain
  native soluble protein

• Learn how to be and
• think like a scientist
• Obtain native
• soluble protein

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Why Do We Study Saccharomyces cerevisiae?

• Model eukaryotic organism
• Grow as haploid or diploid
• Small genome which has
  been sequenced
• Easy to grow, maintain, store
• Non-pathogenic

http//www.nature.com/news/2004/040816/images/yeas
t.jpg
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Haploid yeast of opposite mating type can mate to
form a diploid
a factor
a
a
a factor
a/a diploid
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After stimulation with a factor
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What Does E. Coli Have To Do With It?

• Bacteria that live in mammalian intestine
• Easily grown in the lab
• Capable of high level protein expression

http//www.microbiologyonline.org.uk/ecoli.htm
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Bacterial growth curve

• Organisms introduced into nutrient-rich medium
  grow

• Lag phase

• Exponential phase

• Stationary phase

• Death phase

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Transformation
Taking up of DNA by cells
E. coli cell
pRARE
CmR
Heat and chemical shock Electroparation
KmR
or
AmpR
Transformed cells grow on LB/Cm/Km (orAmp) medium.
Plasmid - Circular piece of DNA.
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pET Expression System
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Gel Electrophoresis

• proteins are unfolded (ie, random coil)
• uniform charge/mass ratio due to SDS
• therefore endogenous charge and shape are not
  major factors
• mobility is inverse of mass
• Stained with Gel Code Blue, general
• Run pre-stained molecular weight marker

Wiser, Mark F, Tulane University
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Wild Wild Westerns

• Use 2 antibodies to attach to 6his tag and make
  fluorescent on the 700-900 nanometer wavelength
  range
• Very selective and sensitive, protein down to
  picogram levels (Coommassie only 50 nanograms of
  protein)

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Lysis

• Used to obtain the maximum soluble fraction of
  our protein
• Applied sonication
• Bug Buster
• Detergent used to facilitate and dislodge the
  protein
• Benzonase and protease inhibitors
• Enzyme that degrades DNA to decrease the
  viscosity of our solution

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Nickel column purification
Nickel Resin
Imidazole
Histidine
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Bio-Rad Protein Assay

• Uses Coomassie Blue.
• Binds to protein - changes color from brown to
  blue.
• Use microplate spectrophotometry and a range of
  proteins of known concentration as standards to
  quantify concentration of solutions.

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Ste11 Putting it All Together
Ashly-Nikkole Davis
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Ste11 Facts

• 717 amino acids
• MW 80,720 Da
• pI 7.19

• Mitogen-activated protein kinase kinase kinase
• Functions in the signaling of pathways that are
  involved in mating and osmoregulation

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Expressing Ste11

• Performed two inductions to express Ste11
• 30 C
• 1 mM IPTG

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The Next Step Lysis
Majority of Ste11 insoluble
Induced Samples
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Tackling Solubility

• At low concentrations, salt slightly increases
  protein solubility
• Goloubinoff P., et al. Chemical Chaperones
  Regulate Molecular Chaperones in Vitro and in
  Cells under Combined Salt and Heat Stresses. The
  Journal of Biological Chemistry 2001 Aug 21 276
  (43) 39586-39591 
• Salt stress
• Accumulation of osmolytes
• Refolding of unfolded polypeptides

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Putting it to the Test Lysis

• Performed lysis under multiple conditions

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Confirming the Results
Performed lysis under conditions that produced
the most soluble protein
S Soluble I Insoluble
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Wrapping it Up

• Induced 1 L sample of protein for purification
• 1 mM IPTG, 30 C

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Fus3 Background Info

• Protein Length 353 amino acids
• Molecular Weight (Da) 40,772
• pI 7.11
• Cellular Location Nucleus, cytoplasm.
• MAP Kinase family.

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Fus3 in the Mating Pathway

• Final kinase in the signal transduction cascade.
• Phosphorylates proteins involved in cell division
  cycle -- stops cell division for mating.
• Phosphorylates proteins involved in transcription.

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Expressing Fus3

200 115 96 51 37 29

• Fus3 easily expressed.
• Induced overnight at 30 C and 1 mM IPTG.
• Next step See whether the protein is soluble.

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Soluble or Insoluble?
A B C D
A Induced, soluble B Uninduced, soluble C
Induced, pellet D Uninduced, pellet

• Induction overnight at 30.
• Most of the protein was insoluble.

Lysis
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Optimizing Fus3 Solubility
Human estrogen receptor expressed in E. coli was
more soluble when 2 ethanol was added during
induction. Nygaard Harlow (2001. Protein
Expr.Purif. 21500-509.)
My Conditions
18 3 Ethanol
30 3 Ethanol
37 3 Ethanol
18
30
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Overnight inductions, with 1 mM IPTG.
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Check for Expression of Fus3
A B C D E
F G
I U I U I U I U
I U I U

• Overnight inductions with 3 ethanol -- hard to
  tell whether there was expression.
• Overnight inductions without ethanol --
  expression, but likely insoluble.

Conditions
B 18 3 Ethanol
C 30 3 Ethanol
D 37 3 Ethanol
E 18
F 30
G 37
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Western Blot
A B C D

• A MW Marker
• B 18 3 Ethanol
• C 18
• D 37

S P S P S P
S soluble P Pellet
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Enhancing Solubility with Ethanol?

• Induction with 3 ethanol for Fus3 at 18, 30,
  and 37
• Induction with 1 and 2 ethanol at 18, 25, 30
  for Ste7.
• Adding ethanol seemed to have little effect on
  protein solubility -- less protein produced, but
  solubility was not increased.

S P
18 3 ethanol Overnight induction
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Solubility
Ind. Insol.
MW Marker
Ind. Soluble
Ind. Whole
Unind. Whole

• (25C)

Overnight induction at 25 C, 1mM IPTG.
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Purification

• Overnight induction at 25 and 1 mM IPTG.
• Nickel-Column purification.
• Took 8 elutions of .5 mL each.

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Was Fus3 purified?

• Ran Elutions 2-5 on gel.
• Elutions werent very clean.
• Is Fus3 there?
• (hard to tell because Adeline, Krystina, and
  Bria seemed to have the same band in their
  elutions).

E2 E3 E4 E5
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Western Blot --gt Fus3
A Flowthrough B Elution 2 C Crude soluble
extract D Crude insoluble extract
A B C D

• There is some Fus3 in the second elution.

28 of the Fus3 produced is soluble using these
conditions (25 C, 1 mM IPTG overnight)
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Ste7(Serine/Threonine MAP kinase kinase)
Required for cell-type-specific transcription and
signal transduction

• amino acids 515
• MW 57,708 Da
• pI 9.92

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Induction
MW U3 I3 UN IN MW
(kDa) 201 115 96 52 38 29

• Induction successful thick bands in induced
  lanes
• 3-hour band thicker than overnight band

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Lysis
MW UW UP US IW IP IS
201 115 96 52 38 29

• Dark band of correct mass appears in induced
  pellet lane
• Ste7 very insoluble

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Varying Temperature IPTG

• Problem Insoluble aggregates

• Proposed solution
• Decrease temperature
• Decrease IPTG concentration

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Varying Temperature IPTG
MW (C) 25 30 18
25 30 18
(mM) 1 0.1 0.01 1 0.1 0.01 1 0.1 0.01 U
U U
100 75 50
W P S
100 75 50
100 75 50
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Varying Temperature IPTG
W P S U

• 25C, 0.1 mM IPTG
• Lower band between 75 kDa and 50 kDa is Ste7
• In pellet lane, degradation evident did not use
  protease inhibitors

250 150 100 75 50
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Taguchi Method

• Engineering method used to improve robustness of
  products
• Want to be sure Ste7 produced
• Generally full factorials used
• 81 experiments
• Taguchi method takes subset
• Increases efficiency significantly
• 9 experiments
• Step 1Choose parameters and levels

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Orthogonal Array
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MW 1S 2S 1P 2P 3S 3P 4S 4P 5S 5P 6S 6P MW 7S
7P MW 8S 8P 9S 9P
201 115 96 52 38 29 20 7
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Quantification of Soluble Protein Produced
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Mean Analysis
A B C D Level 1 1.20 105.57 138.42 172.89 Level
2 200.86 199.83 121.24 95.93 Level
3 215.61 112.29 158.03 148.87 Delta 214.42 94.26 3
6.78 76.96 Rank 1 2 4 3
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Batch Culture Conditions

• 0.2 mM IPTG
• 25C

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Post-purification Confirmation
MW P S FT E3 MW
MW P S FT2 E3 MW

• Ste7 bound to nickel resin but was not eluted off
• Run Western blot with EDTA PBS elution to
  confirm

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Solubility
Ind. Insol.
MW Marker
Ind. Soluble
Ind. Whole
Unind. Whole

• (25C)

Overnight induction at 25 C, 1mM IPTG.
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KSS1
Krystina Daniels
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KSS1 Functions

• Involved in signaling cascade that regulates
  mating and filamentation pathways
• Belongs to MAP Kinase Subfamily along with FUS3
• Located in cytoplasm and nucleus
• Molecular Weight 42,692 (Da)
• pI 5.39

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3hrs.
3hrs.
O/n
O/n
MW




BL21DE3/pRARE-Lac I
3 hr. and overnight Induction at 30C 1mM IPTG
uninduced
induced
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Insoluble
Soluble
Whole
MW
Lysis
BL21DE3/pRARE-Lac I 1mM IPTG
3 hrs. at 30C
uninduced
induced
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3 hrs.
o/n
MW
O/n
MW
3 hrs.
Gel with ER2256 induction
Protein expression for 3hrs./ Overnight induction
37C 1mM IPTG
uninduced
induced
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Western Blot using extracts and whole cells of
ER2256
MW
Whole
Insoluble
Soluble
Mouse Anti-6His Goat Anti-Mouse 800
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Whats Next?

• Obtain soluble protein by using
• different parameters for
• Induction
• -18C, 23C, different
• concentrations of IPTG

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Introducing...
Ste5
Explored by Bria Selhorst
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Lets Understand the Basics

• Ste5 is a kinase cascade scaffold.
• Recruited by the G-beta-gamma subunit, binds to
  subunit and then binds to Ste11, Ste7, Fus3 or
  Kss1.
• Isoelectric Point 5.19
• Molecular Weight (Da) 102,726
• Ste5 is 917 amino acids long.
  

Ste11
Ste5
Ste7
Fus3 or Kss1
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Where to Begin?

1. Transformed BL21pRARELac1 with pET21-MBP-Ste5-6his
   
2. Inoculated, induced at 30C for 3 hours
   overnight, performed lysis, too.
3. Ran in SDS PAGE Gel, and unable to detect Ste5
   expression using Gel Code Blue

Molecular Weights 42,482 Da -MBP 102,726 Da -
Ste5 1,200 Da - 6-his Approx. Total 146,408 Da
A B C D
200
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A molecular weight marker B Induced 3,
insoluble ex. C Induced 3, soluble ex. D
Induced 3, whole cell
96
In kDa
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We Tried 2 Different Routes
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Plasmid Temperature Variation

• Using BL21DE3/pRARELacI, use 2 different
  plasmids. (pET21-MBP-Ste5-6his pET28-Ste5-6his)
• Negative controls induced empty vectors
• Induced 5 hours and overnight on room temperature
  ferris wheel w/ constant IPTG concentration.
• No clear expression using Gel Code Blue.

A B C D E F G H I
200
115
96
5 5 O O 5 5 O
O C M6 C M6 C 6 C 6
C control MMBP 55 hour induction 6 6his
Oovernight
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Use Different Strains

• Tried 3 different Strains.
• BL21DE3/pRARELacI
• -rare tRNAs
• 2. eBL21/DE3 system
• -uses an inhibitor of E. coli RNA polymerase
• 3. ER2256-
• -different genetic background
• ER2256- successful in expression from 30
  overnight induction 1mM IPTG.

200
115
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Finding Optimal Solubility using ER2256
w/pET21MBP-Ste5-6his
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B. C. D. E. F. G. H . I . J. K. L. M.
N.
-Induced 3 hours and Overnight 30C,
Preformed lysis
200 115 96

• Lanes
• A. X
• B. Molecular W. Marker
• C. Whole Cell-Unin, Overnight
• D. Whole Cell- Induced Overnight
• E. X
• F. Soluble Extract-Induced Overnight
• G. X
• H. Insol. Extract- Induced Overnight
• I.X
• J. Whole-Induced 3 hr
• K. X
• L. Soluble-Induced 3 hr
• M. X
• N. Insoluble Extract-3 hr

B. C. D. E. F. G. H. I . J. K. L. M.
N.
200 115 96
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Nickel Column Purification for Ste5
A.
B.
C.
D.
E.

• MW
• B. Insoluble
• C. Soluble
• D. Flow Through
• E. Elution 2

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Western of Nickel Column Purification for Ste5
A.
B.
C.
D.
E.

• MW
• B. Insoluble
• C. Soluble
• D. Flow Through
• E. Elution 2

Band not in the right place according to
molecular weight of Ste5
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In Conclusion

• Ethanol NaCl added during induction have little
  to no effect on protein solubility
• ER2256 is very successful for expressing proteins
• 1 mM and .1 mM of IPTG increase solubility of
  proteins
• 30 may be better for induction than room
  temperature
• Fus3 is most soluble when induced with ImM IPTG
  at 25 C overnight

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And all this would not have been possible were it
not for the muchly appreciated guidance of
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Dr. Evgueny (Jenya) Kroll
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Dr. Myron Williams
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Dr. Joyce Macabéa
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Tonya Love
(self-proclaimed best friend)
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David Pincus
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Dr. Kirsten Benjamin
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Dr. Ian Burbulis(Arent they all so cute and
happy-looking?
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Maybe?(Sorry, Tina. Or rather, thanks for the
photo.)
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Richs Freezer(archaeology practice zone)
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