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Mariana mar2015
- 1. CLONING AND EXPRESSION OF HPLAT VARIANTS IN E.COLI Mariana I. Leรณn Berrรญos Vibha Bansal, Ph. D. University of Puerto Rico at Cayey February 28th, 2015. 1
- 2. OUTLINE โข Introduce proteins of interest: plasminogen activators, and associated problems. โข Discuss previous work. โข Present objectives and plan of research during this semester. 2
- 3. PLASMINOGEN ACTIVATORS (PA) โข Serine proteases PlasminogenPA Plasmin Clot 3
- 4. PLASMINOGEN ACTIVATORS โข Treatments for Cardiovascular diseases โข Leading cause of death in the United States โข Known PAs include tPA, uPA and Streptokinase. http://imgur.com/gallery/v9Yik 4
- 5. TISSUE PLASMINOGEN ACTIVATOR โข Tissue Plasminogen Activator โข Higher Fibrin specificity than uPA and no immunogenicity unlike Streptokinase. โข Nevertheless tPA shows: โข Lack of sufficient fibrin specificity thus leading to side effects โข Low half-life โข 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 โข We developed pHISp2-hPLAT recombinant plasmid and cloned it in E.coli DH5-ฮฑ. โข Mutations of hPLAT gene 8
- 9. ALIGNMENT OF HPLAT VARIANT SEQUENCES โข Multiple Sequence Alignment by CLUSTALW http://www.genome.jp/tools/clustalw/ 9
- 10. OBJECTIVES โข Clone the designed mutant in E. coli BL-21. โข Induce the E. coli cells with IPTG to express the protein of interest. โข Purify the protein by IMAC chromatography. โข Analyze proteinโs: โข Fibrin Specificity โข Half-life โข Inhibition by PAI-1 10
- 11. METHODOLOGY โข Overview: โข Primer Design โข Cloning โข Expression โข Purification โข Analysis 11
- 12. EXPRESSION AND PURIFICATION Liquid culture of Transformed E.coli BL-21 cells Induction with 1.0mM IPTG for 24 hours at 37หC. 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 โข Dr. Vibha Bansal โข Dr. Saurabh Chattopadhyay โข Natalia Espada, Alexandra Rosado, and Jose Javier. โข Danilo Pรฉrez and Abimael Santos โข RISE Program UPR-Cayey โข Ricky Padilla, UPR-Mayagรผez โข 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
- 14. QUESTIONS 14
- 15. CLONING AND EXPRESSION OF HPLAT VARIANTS IN E.COLI Mariana I. Leรณn Berrรญos 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 wasnโt 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 semesterโฆ
- HOW ARE WE GOING TO DO THIS?
- tPA Domainsโฆ
- After the protein is pureโฆ smooth transition
- DH-5alpha have problem with transcription therefore are not used for protein expression, they produce DNA with no mutations. BL-21