Synthesis of recombinant human relaxin H2 in bacteria and its effects on differentiation of carcinoembryonic stem cells
Experiments described in this thesis were designed to develop a reliable method for the synthesis of the recombinant human hormone relaxin H2. Several methods were tested to produce highly pure, biologically active protein quickly, inexpensively and consistently from batch to batch. Relaxin H2 synthesized in bacteria was assessed by SDS-PAGE gel electrophoresis, Western Dot Blotting, Liquid Chromatography/Ultra High Definition Mass Spectrometry, and in vivo blood pressure response experiments using rats. The recombinant relaxin H2 described in this study was compared with commercially available relaxin H2 and was determined to be of equal quality, purity, and biological activity. In addition, to determine whether or not relaxin H2 induced differentiation of murine carcinoembryonic stem cells P19CL6 into cardiomyocytes was compared with a standard methodology using DMSO-induced differentiation. Relaxin H2 induced differentiation but the onset was delayed by four days compared with DMSO treatment. The endpoint of differentiation was determined as the start of spontaneous contractions within clusters of cells in culture. The results revealed that both commercially obtained and bacterially produced relaxin H2 equally caused a four-day delay in the initiation of contractions by cardiac myocytes compared with DMSO-treated controls. Moreover, cDNA microarray analysis of P19CL6 cells in three separate cultures, induced with DMSO (1%, as a control), and experimental treatments with serelaxin (16.7 nM), and bacterially-derived RLN H2 (16.7nM), respectively. There were no differences observed between cell treatments with the serelaxin- and bacterially-derived RLN H2 in the activation/deactivation patterns of 125 affected genes during the progression of the differentiation process.