Date of Award

Fall 2008

Project Type

Thesis

Program or Major

Biochemistry

Degree Name

Master of Science

Abstract

The objective of this research was to develop methods for the production and purification of recombinant lamprey GTH beta. The overall goal was to produce an active purified recombinant lamprey GTH that could be used for immunological, physiological, and histological studies to further understand the role of the gonadotropin in the sea lamprey, Petromyzon marinus. Constructs with different combinations of tethered lamprey gonadotropin beta to human CG alpha and channel catfish gonadotropin alpha were transfected and expressed in stable cell lines of Drosophila (S2) cells and Chinese hamster ovary (CHO) cells. Cell lines were grown at a large scale and protein was collected for purification. Expression of the gonadotropin was tracked through all phases of the scale-up using RT-PCR in conjunction with Western Blot. Initial purification used a combination of nickel sepharose purification for 6XHis tagged product and size exclusion chromatography for untagged products. Activity of concentrated purified fractions was determined through the use of in-vitro culture of lamprey gonadal tissue, as well as in-vitro receptor activity cAMP assays from transiently transfected COS-7 cells. Attempts to combine lamprey GTH beta with other alpha subunits through coexpression and tethered constructs did not increase yield or produce an active product. It is likely that the lamprey GTH alpha will be required in order to conform to the beta subunit for functional protein activity. The results from this study should be used to develop procedures for production of recombinant lamprey gonadotropins and will be especially important once the alpha subunit has been identified.

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