This document outlines a research project to clone and express variants of human plasminogen activator (HPLAT) in E. coli in order to develop an improved tissue plasminogen activator (tPA) with a longer half-life. The researchers designed mutations to the glycosylation sites of tPA and will clone the mutant HPLAT genes into E. coli, induce expression, purify the proteins, and analyze their fibrin specificity, half-life, and inhibition by PAI-1 compared to wild type tPA. The goal is to develop an improved tPA through mutagenesis for treatments of cardiovascular diseases.

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.
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2. OUTLINE
• Introduce proteins of interest: plasminogen
activators, and associated problems.
• Discuss previous work.
• Present objectives and plan of research during this
semester.
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3. PLASMINOGEN ACTIVATORS (PA)
• Serine proteases
PlasminogenPA Plasmin Clot
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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
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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.
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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
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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
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9. ALIGNMENT OF HPLAT
VARIANT SEQUENCES
• Multiple Sequence Alignment by CLUSTALW
http://www.genome.jp/tools/clustalw/
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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
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11. METHODOLOGY
• Overview:
• Primer Design
• Cloning
• Expression
• Purification
• Analysis
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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
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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.
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14. QUESTIONS
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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.
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