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Published on June 1, 2008

Author: Water_Xta1

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Expression and Purification of His 6 -NLS-Cre-MTS Protein from E. coli BL21(DE3) Heather Jordan Department of Biochemistry and Molecular Biology The Pennsylvania State University University Park, Pennsylvania 16802 December 10, 2002 http://www.ozfactors.com/PICTURES/PSYCHS/Psychiatry.html

What is His 6 - NLS -Cre- MTS ? A recombinant cell-permeable Cre protein His 6 affinity tag Binds to resin (part of purification process) Nuclear Localization Signal Located anywhere in the primary sequence of the protein Directs the protein for transport through the nuclear pore complex (from cytosol to nucleus) Membrane Translocation Sequence Directs protein into the cell (from cell surface to cytosol)

A recombinant cell-permeable Cre protein

His 6 affinity tag

Binds to resin (part of purification process)

Nuclear Localization Signal

Located anywhere in the primary sequence of the protein

Directs the protein for transport through the nuclear pore complex (from cytosol to nucleus)

Membrane Translocation Sequence

Directs protein into the cell (from cell surface to cytosol)

Cre protein is… A member of the integrase family (catalyzes an insertion reaction in DNA) Encoded by the E. coli phage P1 During cell division, Cre separates dimers of P1 that arise after replication

A member of the integrase family (catalyzes an insertion reaction in DNA)

Encoded by the E. coli phage P1

During cell division, Cre separates dimers of P1 that arise after replication

Cre protein is… A member of the integrase family (catalyzes an insertion reaction in DNA) Encoded by the E. coli phage P1 During cell division, Cre separates dimers of P1 that arise after replication

A member of the integrase family (catalyzes an insertion reaction in DNA)

Encoded by the E. coli phage P1

During cell division, Cre separates dimers of P1 that arise after replication

Cre protein is… A member of the integrase family (catalyzes an insertion reaction in DNA) Encoded by the E. coli phage P1 During cell division, Cre separates dimers of P1 that arise after replication Replication

A member of the integrase family (catalyzes an insertion reaction in DNA)

Encoded by the E. coli phage P1

During cell division, Cre separates dimers of P1 that arise after replication

Cre protein is… A member of the integrase family (catalyzes an insertion reaction in DNA) Encoded by the E. coli phage P1 During cell division, Cre separates dimers of P1 that arise after replication Homologous Recombination Replication

A member of the integrase family (catalyzes an insertion reaction in DNA)

Encoded by the E. coli phage P1

During cell division, Cre separates dimers of P1 that arise after replication

Cre protein is… A member of the integrase family (catalyzes an insertion reaction in DNA) Encoded by the E. coli phage P1 During cell division, Cre separates dimers of P1 that arise after replication Cre-mediated recombination Homologous Recombination Replication Modified from http://www.esb.utexas.edu/jayaramlab/recomb/Cre.html

A member of the integrase family (catalyzes an insertion reaction in DNA)

Encoded by the E. coli phage P1

During cell division, Cre separates dimers of P1 that arise after replication

How does Cre work? Site of action is the loxP site. loxP consists of two 13 bp inverted binding sites separated by a 6 bp spacer . Cleavage sites loxP site

Site of action is the loxP site.

loxP consists of two 13 bp inverted binding sites separated by a 6 bp spacer .

Cleavage sites

How does Cre work? Model of Recombination: Cre reacts with a 6 bp spacer and cleaves in cis at the arrowheads. Proteins bring the DNA site together in an antiparallel synapse with the green monomers active and the blue monomers supressed.

Model of Recombination:

How does Cre work? Model of Recombination: Strand swapping occurs and the Holliday junction intermediate is formed.

Model of Recombination:

How does Cre work? Model of Recombination: The proteins undergo an allosteric conformational change. Now, the blue monomers are active for cleavage and the green ones are supressed.

Model of Recombination:

How does Cre work? Model of Recombination: Modified from http://www.esb.utexas.edu/jayaramlab/model.html

Model of Recombination:

Study Objective In floxed (fg2) mice the addition of the protein pops out the gene coding for the function of axon 8 in the  -2 subunit. Can be used selectively to knock out this gene in different regions of the brain http://web1.nsac.ns.ca/news/ac_post/1997/aug97/mouse. htm

In floxed (fg2) mice the addition of the protein pops out the gene coding for the function of axon 8 in the  -2 subunit.

Can be used selectively to knock out this gene in different regions of the brain

Why knock out  -2 this way? Current Method: Cross-breeding New Method: Protein Injection EMX-1

Materials and Methods Expression Growing the E. coli Induce over expression with IPTG Freezing the cells Purification Sonication & centrifugation Ni-NTA resin & elution Preparation Filter Dialysis With aCSF Quantitation Bradford Assay

Expression

Growing the E. coli

Induce over expression with IPTG

Freezing the cells

Purification

Sonication & centrifugation

Ni-NTA resin & elution

Preparation

Filter

Dialysis

With aCSF

Quantitation

Bradford Assay

Materials and Methods Expression Growing the E. coli Induce over expression with IPTG Freezing the cells Purification Sonication & centrifugation Ni-NTA resin & elution Preparation Filter Dialysis With aCSF Quantitation Bradford Assay

Expression

Growing the E. coli

Induce over expression with IPTG

Freezing the cells

Purification

Sonication & centrifugation

Ni-NTA resin & elution

Preparation

Filter

Dialysis

With aCSF

Quantitation

Bradford Assay

Precipitate?! That’s not supposed to be there! Protein crashed out Why? High [salt] pH How do we find out what’s wrong? Recalculated protocol amounts (esp. hydrated salts) pH individual components of aCSF Test every 2 components (of the 3) against each other to see if ppt. forms

That’s not supposed to be there!

Why?

High [salt]

pH

How do we find out what’s wrong?

Recalculated protocol amounts (esp. hydrated salts)

pH individual components of aCSF

Test every 2 components (of the 3) against each other to see if ppt. forms

Results of 2-Component Test aCSF alone = CLEAR aCSF + MgCL2 = CLEAR aCSF + CaCl2 = WHITE PPT.       Is there another protocol for aCSF that might work better?

Artificial Cerebrospinal Fluid Formulations NaCl (124 mM) KCl (1.6 mM) Glucose CaCl 2 (2.5 mM) MgCl 2 (1.5 mM) NaHCO 3 (24 mM) KH 2 PO 4 (1.2 mM) Ascorbic Acid (2 mM) NaCl (147 mM) KCl (2.9 mM) Dextrose CaCl 2 (1.7 mM) MgCl 2 (1.6 mM) First, we tried: Then, we tried:

NaCl (124 mM)

KCl (1.6 mM)

Glucose

CaCl 2 (2.5 mM)

MgCl 2 (1.5 mM)

NaHCO 3 (24 mM)

KH 2 PO 4 (1.2 mM)

Ascorbic Acid (2 mM)

NaCl (147 mM)

KCl (2.9 mM)

Dextrose

CaCl 2 (1.7 mM)

MgCl 2 (1.6 mM)

Artificial Cerebrospinal Fluid Formulations NaCl ( 124 mM ) KCl ( 1.6 mM ) Glucose CaCl 2 ( 2.5 mM ) MgCl 2 ( 1.5 mM ) NaHCO 3 (24 mM) KH 2 PO 4 (1.2 mM) Ascorbic Acid (2 mM) NaCl ( 147 mM ) KCl ( 2.9 mM ) Dextrose CaCl 2 ( 1.7 mM ) MgCl 2 ( 1.6 mM ) First, we tried: Then, we tried:

NaCl ( 124 mM )

KCl ( 1.6 mM )

Glucose

CaCl 2 ( 2.5 mM )

MgCl 2 ( 1.5 mM )

NaHCO 3 (24 mM)

KH 2 PO 4 (1.2 mM)

Ascorbic Acid (2 mM)

NaCl ( 147 mM )

KCl ( 2.9 mM )

Dextrose

CaCl 2 ( 1.7 mM )

MgCl 2 ( 1.6 mM )

Artificial Cerebrospinal Fluid Formulations NaCl ( 124 mM ) KCl ( 1.6 mM ) Glucose CaCl 2 ( 2.5 mM ) MgCl 2 ( 1.5 mM ) NaHCO 3 (24 mM) KH 2 PO 4 (1.2 mM) Ascorbic Acid (2 mM) NaCl ( 147 mM ) KCl ( 2.9 mM ) Dextrose CaCl 2 ( 1.7 mM ) MgCl 2 ( 1.6 mM ) First, we tried: Then, we tried:

NaCl ( 124 mM )

KCl ( 1.6 mM )

Glucose

CaCl 2 ( 2.5 mM )

MgCl 2 ( 1.5 mM )

NaHCO 3 (24 mM)

KH 2 PO 4 (1.2 mM)

Ascorbic Acid (2 mM)

NaCl ( 147 mM )

KCl ( 2.9 mM )

Dextrose

CaCl 2 ( 1.7 mM )

MgCl 2 ( 1.6 mM )

Artificial Cerebrospinal Fluid Formulations NaCl ( 124 mM ) KCl ( 1.6 mM ) Glucose CaCl 2 ( 2.5 mM ) MgCl 2 ( 1.5 mM ) NaHCO 3 (24 mM) KH 2 PO 4 (1.2 mM) Ascorbic Acid (2 mM) NaCl ( 147 mM ) KCl ( 2.9 mM ) Dextrose CaCl 2 ( 1.7 mM ) MgCl 2 ( 1.6 mM ) First, we tried: Then, we tried:

NaCl ( 124 mM )

KCl ( 1.6 mM )

Glucose

CaCl 2 ( 2.5 mM )

MgCl 2 ( 1.5 mM )

NaHCO 3 (24 mM)

KH 2 PO 4 (1.2 mM)

Ascorbic Acid (2 mM)

NaCl ( 147 mM )

KCl ( 2.9 mM )

Dextrose

CaCl 2 ( 1.7 mM )

MgCl 2 ( 1.6 mM )

Artificial Cerebrospinal Fluid Formulations NaCl (124 mM) KCl (1.6 mM) Glucose CaCl 2 (2.5 mM) MgCl 2 (1.5 mM) NaHCO 3 (24 mM) KH 2 PO 4 (1.2 mM) Ascorbic Acid (2 mM) NaCl ( 147 mM ) KCl ( 2.9 mM ) Dextrose CaCl 2 ( 1.7 mM ) MgCl 2 ( 1.6 mM ) First, we tried: Then, we tried:

NaCl (124 mM)

KCl (1.6 mM)

Glucose

CaCl 2 (2.5 mM)

MgCl 2 (1.5 mM)

NaHCO 3 (24 mM)

KH 2 PO 4 (1.2 mM)

Ascorbic Acid (2 mM)

NaCl ( 147 mM )

KCl ( 2.9 mM )

Dextrose

CaCl 2 ( 1.7 mM )

MgCl 2 ( 1.6 mM )

How was this conclusion reached? Had the same problem making anaerobic media The NaHCO 3 was identified as the ‘problem ingredient’ Also yielded white precipitate New buffer system devised (using KH 2 PO 4 and K 2 HPO 4 instead of NaHCO 3 )

Had the same problem making anaerobic media

The NaHCO 3 was identified as the ‘problem ingredient’

Also yielded white precipitate

New buffer system devised (using KH 2 PO 4 and K 2 HPO 4 instead of NaHCO 3 )

Other Problems Encountered Shaker temperature Changed from 37 o C to 25 o C. Centrifuge noise Tubes not swinging freely Slight imbalance

Shaker temperature

Changed from 37 o C to 25 o C.

Centrifuge noise

Tubes not swinging freely

Slight imbalance

Results

Results Band A : 75.148x -1.1081 = 1.3  x = 38.9 kDa Band B : 75.148x -1.1081 = 2.3  x = 23.25 kDa Band C : 75.148x -1.1081 = 2.7  x = 20.12 kDa Desired Protein Product Possible Protein Degradation Products  A   B   C  Ladder Pst-son Pst-B2 Pst-B3 Flo-Th Final 1.3  2.3  2.7 

Band A : 75.148x -1.1081 = 1.3  x = 38.9 kDa

Band B : 75.148x -1.1081 = 2.3  x = 23.25 kDa

Band C : 75.148x -1.1081 = 2.7  x = 20.12 kDa

Future Directions Most protein loss occurred early in protocol (before first aliquot) Try it again with the following changes: Less sonication (extra bands are probably degradation products of desired protein) Monitor sonication more closely (checking viscosity) Leave purification steps unchanged (no significant contamination in aliquots)

Most protein loss occurred early in protocol (before first aliquot)

Try it again with the following changes:

Less sonication (extra bands are probably degradation products of desired protein)

Monitor sonication more closely (checking viscosity)

Leave purification steps unchanged (no significant contamination in aliquots)

Acknowledgements Clint Earnheart Everyone else in the L ü scher lab http://www.criver.com/xruk/transgenics.html - I’m chimeric but I’m cute!

Clint Earnheart

Everyone else in the L ü scher lab

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