Initial study of truncated human β-tropomyoson with mutations related to neromuscular disorders

Abstract

This report regards an initial part of a larger study aiming towards structure determination of six β-tropomyosin mutations, mapped during clinical investigations of congenital myopathies performed at Sahlgrenska University hospital. This initial project includes methods and suggestions as to how the tropomyosin protein molecule can be truncated to produce a suitable platform for high resolution structure determination, using x-ray crystallography. During this study Ligation Independent Cloning, LIC, was evaluated as cloning method, and this method is discussed with practical conclusions presented in the end of the report. For the tropomyosin truncation design a thorough literature study was conducted, over viewing all presently published material of x-ray crystallography regarding tropomyosin. Cloning and vector amplification were performed in Escherichia coli. This study points out the necessity of making a thorough investigation of present information to be able to deduce protein truncations, which not only will have good prerequisites for high resolution structure determination, but also include as many intact interaction sites with other sarcomeric proteins as possible. Also, this study shows that LIC can be an efficient and high throughput method for cloning gene fragments.