Acquisition And Identification Of Nacreous Proteins And Their Interactions With Calcium Ions

Nacreous layers in mollusk shells are formed by the inducing and medicating of nacreous proteins under the mechanism of biomineralization. The study of nacreous proteins is not only essential to understand the formation of pearl and shell but also important for understanding the mechanism of biomineralization. Proteins are extracted from nacreous layers of Pinctada fucata shells with acid-dissolving method, after which they are purified and identified. Heterologous expressions of two specific proteins are carried out respectively. What’s more, the nacreous proteins are loaded on hybrid films of gold nanoparticles and cellulose. And the interaction between the proteins and Ca²⁺ is also involved.Extracted proteins are examined with SDS-PAGE and MS. It is shown that acid-dissolving proteins are in accordance with the nacreous proteins discovered in Pinctada fucata. Three novel proteins in nacre are purified by HPLC and identified with MS.Two kinds of nacreous protein, Nacrein and N16, are expressed in E. coli. It is found that Nacrein finally forms non-soluable inclusion bodies with a low expression amount. Denatured Nacrein is purified by affinity chromatography method and then identified with Western Blot. Largely expressed N16 is partly soluble, making it easy to be purified and applied to following tests.Hybrid films of gold nanoparticles and cellulose with high gold contents are prepared and used as electrochemical electrodes to study interactions between relevant proteins and Ca²⁺. Firstly, the feasibility of making use of hybrid films as electrochemical electrodes is verified with CV and EIS methods. Secondly, BSA and Ca M are loaded on hybrid films seperatedly to test the loading status of BSA and the interaction with Ca²⁺ of Ca M with EIS methods respectively. Then, extracted nacreous proteins and expressed N16 are loaded on hybrid films seperatedly. It is found that interactions between these proteins and Ca²⁺ are very significant and can be differentiated from the impedence responses in high frequence regions. This phenomenon is very important for the recognition of biomineralization proteins from shells with electrochemical methods.