Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2020991 | Protein Expression and Purification | 2010 | 5 Pages |
Abstract
Mutations in the fibrinogen gene cluster can cause low plasma fibrinogen concentrations, known as hypofibrinogenaemia. It is important to verify whether a detected sequence variant in this cluster is deleterious or benign and this can be accomplished using protein expression systems. In this study, four mutations in the fibrinogen γC domain that had previously been described in patients with hypofibrinogenaemia were introduced into a γC construct and expressed in a Pichia pastoris yeast system to investigate their effects on protein stability and secretion. These experiments showed that the fibrinogen Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y), and Muncie (T371I) mutations were not secreted, supporting their causative role in hypofibrinogenaemia. Overexpression of the N230D, A289V and H307Y mutants revealed that the majority of the synthesised protein was retained in the endoplasmic reticulum, with only a minor proportion reaching the trans-Golgi network. Regardless, none of this protein was secreted which confirms that the four mutations investigated are indeed responsible for hypofibrinogenaemia.
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Campbell R. Sheen, Amy Dear, Stephen O. Brennan,