Juliet McGill

Senior Project Advisor

P. Clint Spiegel

Document Type

Project

Publication Date

Fall 2023

Keywords

Blood coagulation, Factor IX, Factor IX mutants, Factor VIII, Inclusion Bodies, Protein Purification, Refolding Protein, Activation of FIX with FXIa, Protein Crystallization

Abstract

This paper presents readers with an optimized procedure for the purification, activation, and crystallization of selected blood coagulation Factor IX double mutant (FIX_2). Through the completion of this work, we aim to enhance future biochemical and structural studies by providing an easier means for the FIX_2 production, in order to increase understanding of the protein’s function within the blood coagulation cascade. The initiation of the blood coagulation cascade is brought on by activation of inactive Factor VIII (FVIII) protein though contact with tissue factor, the FVIII protein then binds to an activated platelet surface where it must wait for its serine protease cofactor, Factor IX (FIX)1. Just as many other coagulation factors, FIX is produced and circulating in its inactive, catalytically silent form and must be activated by another protein Factor XIa, through cleavage of the amino acid sequence known as the activation peptide 2, 3. Once both proteins have become activated, the platelet bound active FVIII (FVIIIa) coordinates with active FIX (FIXa) to form the potent Xase complex 1, 2. This Xase complex formation results in structural rearrangement of the FIX’s 99-loop and 60-loop to permit the proteolytic cleavage action of FIXa on Factor X (FX) converting it into active FX (FXa) 4. This FXa protein is subsequently used for the activation of thrombin which heavily induces clot formation 3. However, structural understanding of the assembly and activity of the Xase complex is still relatively unknown therefore the study of the Xase complex and its operating co-factors, FVIII and FIX, remains a highly active field of research.

Department

Chemistry

Subjects - Topical (LCSH)

Blood--Coagulation; Erythrocytes--Inclusions; Molecular biology

Type

Text

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Language

English

Format

application/pdf

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