Date of Award

Fall 1999

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Laura K. Moen

Committee Member

Mark S. Elliot

Committee Member

James H. Yuan

Call Number for Print

Special Collections LD4331.C45 K457

Abstract

Human T-cell leukemia virus type 1 (HTL V-1) is dependent upon the enzymatic activity of its protease for maturation. Maturation of the protease is facilitated by cleavage of specific amino acid residues, followed by dimerization. The effects of the amino acid sequence located N-terminally to the cleavage site on the ability of the protease to become active were the focus of the current study. These amino acid sequences were contributed by the plasmid vector into which the protease gene was inserted.

Surface probability analyses (SPAs) of the vectors, as well as for native sequences which produce the mature protease and other viral proteins indicated that a specific surface topology may be required for cleavage site recognition by the protease. Using this information, two plasmid vectors, pETl 5b and pETl 9b, were selected for use. SPA of the pET15b vector indicated that amino acid sequence located N-terminally to the cleavage site was hydrophobic. In the pETl 9b vector, the sequence was hydrophilic. Three clones of the protease gene were synthesized and inserted into each vector, leading to six protease constructs.

The protease gene was expressed, purified, and activated in each of the constructs. The enzyme was activated against two different buffer systems. One buffer contained sodium citrate, an anti-chaotroph. The other contained 2-( 4-morpholino) ethanesulfonic acid (MES). The use of two different buffer systems provided the opportunity to assess the effects of buffers on the activation process. Fluorometry was used to evaluate enzymatic activity.

Results of this study indicated that both the N-terminal amino acid sequence and the composition of the buffer against which the enzyme was activated may affect the level of enzyme activation in specific constructs. Protease constructs composed of a protease gene inserted into the pETl 5b vector exhibited two to four times more enzymatic activity than the same gene inserted into the pETl 9b vector. The use of sodium citrate to promote activation resulted in higher activity levels than when MES buffer was used for activation. The increased level of activation when citrate buffer was used ranged from approximately 1 ¼ times to almost three times the level of activity seen when MES buffer was used.

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DOI

10.25777/dnbv-cs29

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