Date of Award:

2013

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Advisor/Chair:

Randolph V Lewis

Abstract

Kisspeptins are an arginine-phenylalanine-amide family of neuroactive peptide hormones encoded by the KISS1 gene, which, along with its receptor GPR54, are essential for the initiation of puberty in adolescents and maintenance of reproductive function in adults. Kisspeptins are also involved in the sexual dimorphism of the brain. Kisspeptin signaling defects are implicated in reproductive disorders like idiopathic hypogonadotropic hypogonadism, precocious puberty and polycystic ovarian syndrome. Kisspeptins were originally discovered as metastasis suppressors in melanoma cell lines and have also been found to down regulate metastasis in breast and ovarian cancers. They have been found to inhibit the colonization of secondary tissues by the metastatic cancer cells. Kisspeptins also relay nutritional status to the reproductive system. However, little is known about the mechanism of action of kisspeptins in a cellular and biochemical context. Therefore, an important goal is the synthesis of kisspeptins in sufficient quantities for use as experimental and therapeutic reagents. In this study, we developed a simple and cost-effective method to express and purify kisspeptins in large quantities. We designed the kisspeptin constructs in-silico and expressed them in an Escherichia coli bacterial system. The purification was then optimized using standard chromatographic techniques. In anticipation of future demands, we successfully scaled the expression and purification of the kisspeptins using larger bioreactors and chromatography systems. We tested the biological activity of the kisspeptin we produced on mammalian cell lines expressing the kisspeptin receptor and compared its actions to the kisspeptins synthesized by chemical means. We found that kisspeptins activated two proteins involved in the cell proliferation underlying metastasis. Thus, we were able to show that the much lower cost kisspeptins synthesized by Escherichia coli act similarly to proteins manufactured by chemical synthesis.

Included in

Biology Commons

Share

COinS