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Mariana mar2015
1. CLONING AND EXPRESSION OF
HPLAT VARIANTS IN E.COLI
Mariana I. León Berríos
Vibha Bansal, Ph. D.
University of Puerto Rico at Cayey
February 28th, 2015.
1
2. OUTLINE
• Introduce proteins of interest: plasminogen
activators, and associated problems.
• Discuss previous work.
• Present objectives and plan of research during this
semester.
2
4. PLASMINOGEN ACTIVATORS
• Treatments for Cardiovascular
diseases
• Leading cause of death in the
United States
• Known PAs include tPA, uPA and
Streptokinase.
http://imgur.com/gallery/v9Yik
4
5. TISSUE PLASMINOGEN ACTIVATOR
• Tissue Plasminogen Activator
• Higher Fibrin specificity than uPA and no
immunogenicity unlike Streptokinase.
• Nevertheless tPA shows:
• Lack of sufficient fibrin specificity thus
leading to side effects
• Low half-life
• Sensitivity to inhibition by PAI-1.
5
6. TPA STRUCTURE
Domain Residues Function
Finger 4-50 Fibrin Specificity
Epidermal Growth
Factor
51-87 Protein secretion
Kringle 1 88-175 ???
Kringle 2 176-256 Fibrin Binding,
Glycosylation sites
Protease 257-527 Enzymatic Activity,
Glycosylation Sites
An improved tPA, with longer half-life can be obtained
through mutagenesis!
http://www.chem.cmu.edu/groups/Llinas/res/structure/tpa-big.html
6
7. 7
NH2
COOHF E K1 K2 P
50 87 175 256 527
Asn184
Asn448
Asn117
Smaller protein is better expressed!
NH2 COOHK2 P
175 256 527
Asn448Asn184
Mutate first Glycosylation site
NH2 COOHK2 P
175 256 527
Asn448Gln184
Mutate second Glycosylation site
NH2 COOHK2 P
175 256 527
Gln448Gln184
M1
M2
M3
WtMutation Design
8. PREVIOUS WORK
• We developed pHISp2-hPLAT recombinant plasmid
and cloned it in E.coli DH5-α.
• Mutations of hPLAT gene
8
9. ALIGNMENT OF HPLAT
VARIANT SEQUENCES
• Multiple Sequence Alignment by CLUSTALW
http://www.genome.jp/tools/clustalw/
9
10. OBJECTIVES
• Clone the designed mutant in E. coli BL-21.
• Induce the E. coli cells with IPTG to express
the protein of interest.
• Purify the protein by IMAC chromatography.
• Analyze protein’s:
• Fibrin Specificity
• Half-life
• Inhibition by PAI-1
10
12. EXPRESSION AND PURIFICATION
Liquid culture of
Transformed
E.coli BL-21 cells
Induction with
1.0mM IPTG
for 24 hours at 37˚C.
Cell lysis using lysis
buffer
Purification using
Immobilized Metal Ion
Affinity
Chromatography
Miniprep product
Transformation
of BL-21
E. coli cells
Pure Protein for
characterization
12
13. ACKNOWLEDGEMENTS
• Dr. Vibha Bansal
• Dr. Saurabh Chattopadhyay
• Natalia Espada, Alexandra Rosado, and Jose Javier.
• Danilo Pérez and Abimael Santos
• RISE Program UPR-Cayey
• Ricky Padilla, UPR-Mayagüez
• National Center for Research Resources and the
National Institute of General Medical Sciences of the
National Institutes of Health through Grant Number 8
P20 GM 103475.
13
15. CLONING AND EXPRESSION OF
HPLAT VARIANTS IN E.COLI
Mariana I. León Berríos
Vibha Bansal, Ph. D.
University of Puerto Rico at Cayey
February 28th, 2015.
15
Editor's Notes
There are three common PAs
tPA: - Hydrolyzes ARG-VAL bond in plasminogen STREPTOKINASE-from BACTERIA!! Not Human!!!
-Second generation Plasminogen Activator
-Composed of 562 Amino Acids and 5 domains
Domains: The term domain was defined by Jane Richardson in 1981 as a part of a polypeptide chain that is independently stable or could undergo movements as a single entity with respect to the entire protein.
Although we expressed the wt-tpa it wasn’t so active because it was too big for the bacteria to express it. The deletions helped the process because in bacteria smaller proteins are better expressed and hence the proteins obtained were REALLY active. The bacteria cloning and expressing system was used because they were less complicated than mammals cells and
So for this semester…
HOW ARE WE GOING TO DO THIS?
tPA Domains…
After the protein is pure… smooth transition
DH-5alpha have problem with transcription therefore are not used for protein expression, they produce DNA with no mutations.
BL-21
