Preparation and Evaluation of Recombinant Statherin

Abstract

Introduction: Statherin is a 43-residue peptide encoded by a single copy gene (SATH gene) located inside chromosome 4q11-13. Statherin has a low molecular weight (Mr = 5380). It is secreted from parotid and submandibular salivary glands. Statherin is a multifunctional molecule, variety of action has been linked to statherin such as having a high affinity for calcium phosphate mineral such as hydroxyapatite (HAP). Statherin is a strong inhibitor to calcium phosphate precipitation despite being in a supersaturated condition. Statherin prevent both crystal growth and spontaneous precipitation, thus allowing calcium and phosphate to be readily available to be utilised by enamel re-calcification and stabilisation as well as, inhibiting the development of mineral accretion on the tooth surface. The aim of this research is to produce recombinant statherin in a bacterial expression system and to introduce phosphorylation mimicking aspartic acid at potential phosphorylation site serine at position 1 and 2 and to evaluate biological properties of the various recombinant statherin.

Methods: Gene cloning and activation through using pET32a vector in NiCo21 (DE3) competent E. coli, pET32a vector in BL21 (DE3) pLysS competent E. coli. And Gibson Assembly using NEB 5-alpha Competent E. coli, with pCHAPAd4 vector.

Results: ELISA activity detected with pET32a vector in BL21 (DE3) pLysS competent E. coli. Western bolting revealed band presence, however not in the desired location.

Conclusions: Based on the activity observed with ELISA, protein of interest (statherin) is produced, it is seen with all variants of statherin DD, DS, SD, and SS, however further optimisation is needed for sufficient protein for further analysis.