Identifier

etd-04272011-093156

Degree

Master of Natural Sciences (MNS)

Department

Natural Sciences (Interdepartmental Program)

Document Type

Thesis

Abstract

Erythropoietin (EPO) is the main cytokine regulator for red blood-cell production in the human by binding to erythropoietin receptors to promote erythroid proliferation, survival and differentiation. Administration of recombinant human EPO (rhEPO) as a therapeutic has been shown to improve renal and non-renal anemia and this success has led to a high medical global demand. Chinese hamster ovary (CHO) cells, yeast, and bacteria have been used to produce rhEPO but nevertheless there is still a need for a low-cost and high quality expression system for rhEPO. LMH/2A cells, as an alternative model, were transfected with the codon optimized human EPO gene for glycosylated rhEPO production. The LMH/2A-rhEPO was detected and purified using a series of chromatographic steps and the purity was determined to be 95%. As expected, LMH/2A-rhEPO was N-linked glycosylated indicated by a considerable shift in molecular weight after enzymatic deglycosylation with PNGaseF. The biological activity of purified LMH/2A-rhEPO was investigated in vitro using cell based assay (CD34+ cells) and was compared to the biological activities of rhEPO expressed from CHO cells and human cells (HEK293). In this study, LMH/2A-rhEPO induced cell proliferation which showed a five fold increase in the number of rhEPO-treated CD34+ cells as compared to the number of non-treated CD34+ cells. Furthermore, rhEPO-treated CD34+ cells expressed hemoglobin 2500 fold higher than non-treated CD34+ cells. Growing CD34+ cells supplemented with LMH/2A-rhEPO resulted in an increase in the expression of erythroid markers such as transferrin receptors, glycophorin A, and hemoglobin. Interestingly, EPO with LMH/2A cell specific glycosylation promotes similar erythroid differentiation of human CD34+ cells, compared to CHO and human cell expressed rhEPO. Therefore, LMH/2A-rhEPO was biologically active in vitro by inducing erythroid differentiation which indicates that LMH/2A cells can definitely can be used as a suggested alternative system for rhEPO production.

Date

2011

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Cooper, Richard

DOI

10.31390/gradschool_theses.156

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