Expression and Purification of Human Platelet-Derived Growth Factor Beta (Pdgf-B)

Topics: Molecular biology, DNA, Bradford protein assay Pages: 10 (3122 words) Published: January 13, 2013
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 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. Thus, these isoforms combine with each other by forming homodimers or heterodimers bound through disulfide bonds [1, 3]. Three active receptors for PDGF included PDGFRα, PDGFR-β and PDGFR-αβ (Figure I) has been shown to be specific for the PDGF dimers [3]. The extracellular region of the receptor consists of five immunoglobulin-like domains while the intracellular part is a tyrosine kinase domain [1-3]. The activation of these receptors conducts to a larger propagation of the signal [3].

Figure I. Protein recognition by the PDGF receptors PDGFR-α, PDGFR-β and PDGFR-αβ [3]. Human PDGF-BB is homodimeric, not glycosilated and is mostly expressed in endothelial vascular cells [4]. Its polypeptide chain contains 109 amino acids and has a molecular weight of 24.3 kDa. The peptide chain of PDGF-BB contains a basic domain highly conserved among the growth factors [4].


Final Report Submission

BIND 5005-Fall 2009

The main goal of this research project is to synthesize the recombinant human PDGF-B isomer on a bacterial host for its isolation and purification. The PDGF-B gene will be expressed by transformation of competent KRX cells with the expression vector called pReceiver-B04. KRX is a modified strain of E. coli (K12) that has associated applications with cloning, screening strains and engineering attributes for protein expression optimization [5]. It incorporates a chromosomal copy of polymerase RNA T7 guided by the rhamnose promoter (rhapBAD) to provide a dramatic control of recombinant protein expression. The RNA T7 polymerase and its promoter show its elongation time at a fast rate (up to 5 times more fast that E. coli’s RNA polymerase) and it is completely independent from E. coli’s RNA polymerase receptors. RNA T7 polymerase replaces rhaPBAD in order for the expression to be controlled by its promoter. pReceiver-B04 has a promoter for the RNA T7 polymerase; its host cell is E. coli; has an antibiotic resistance for ampicillin; and contains a...
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