Expression and Purification of Human Platelet-Derived Growth Factor Beta (Pdgf-B)
Expression and Purification of Human Platelet-Derived Growth Factor Beta (PDGF-B)
Rachel C. Hermina-Stewart* and Elsie I. Parés-Matos† *Industrial Biotechnology Program, †Department of Chemistry University of Puerto Rico-Mayagüez Campus rachel.hermina@upr.edu December 14, 2009 ABSTRACT. Platelet-Derived Growth Factor (PDGF) is one of many proteins that regulate cellular growth and division. The PDGF family consists mainly of five different isoforms called as PDGF-A, PDGF-B, PDGF-C and PDGF-D. Three active receptors for PDGF include PDGFR-α, PDGFR-β and PDGFRαβ, have been shown to be specific for the PDGF dimers. PDGF-B becomes active when dimeric and it is recognized by the three PDGF receptors. PDGF has served as a study model for the recognition of the nucleotide sequence of an aptamer. High levels of PDGF are indicative of abnormal cellular growth (conglomerates, cancer). Preliminary clinical studies show that PDGF-B levels can be monitored on the bloodstream and, in contrast from its fellow isoforms, it has the ability to activate each of the PDGF receptors (-α, -β and -αβ) shifting the balance of signaling to favor the transformation pathway. This research aims to express and purify the recombinant human PDGF-B in order to study its properties with a selected aptamer. The expression of the PDGF-B gene, cloned in the expression vector pReceiver-B04, will be controlled with rhamnose in a bacterial system suitable for the RNA T7 polymerase. The recombinant protein will be isolated, purified and characterized by SDS-PAGE and Western blot. Several chromatographic techniques including gel filtration and affinity chromatography will also be employed for the purification of the recombinant human PDGF-B.
Final Report Submission
BIND 5005-Fall 2009
INTRODUCTION. The PDGF family consists mainly of four isoforms called as PDGF-A, PDGF-B, PDGF-C and PDGF-D, where the last two being the newly discovered [1-3]. The four PDGFs are inactive in their monomeric form.
Rachel C. Hermina-Stewart* and Elsie I. Parés-Matos† *Industrial Biotechnology Program, †Department of Chemistry University of Puerto Rico-Mayagüez Campus rachel.hermina@upr.edu December 14, 2009 ABSTRACT. Platelet-Derived Growth Factor (PDGF) is one of many proteins that regulate cellular growth and division. The PDGF family consists mainly of five different isoforms called as PDGF-A, PDGF-B, PDGF-C and PDGF-D. Three active receptors for PDGF include PDGFR-α, PDGFR-β and PDGFRαβ, have been shown to be specific for the PDGF dimers. PDGF-B becomes active when dimeric and it is recognized by the three PDGF receptors. PDGF has served as a study model for the recognition of the nucleotide sequence of an aptamer. High levels of PDGF are indicative of abnormal cellular growth (conglomerates, cancer). Preliminary clinical studies show that PDGF-B levels can be monitored on the bloodstream and, in contrast from its fellow isoforms, it has the ability to activate each of the PDGF receptors (-α, -β and -αβ) shifting the balance of signaling to favor the transformation pathway. This research aims to express and purify the recombinant human PDGF-B in order to study its properties with a selected aptamer. The expression of the PDGF-B gene, cloned in the expression vector pReceiver-B04, will be controlled with rhamnose in a bacterial system suitable for the RNA T7 polymerase. The recombinant protein will be isolated, purified and characterized by SDS-PAGE and Western blot. Several chromatographic techniques including gel filtration and affinity chromatography will also be employed for the purification of the recombinant human PDGF-B.
Final Report Submission
BIND 5005-Fall 2009
INTRODUCTION. The PDGF family consists mainly of four isoforms called as PDGF-A, PDGF-B, PDGF-C and PDGF-D, where the last two being the newly discovered [1-3]. The four PDGFs are inactive in their monomeric form.