BSGC Role

Dr. Rosalind Kim is a molecular biologist who has been developing and testing the heterologous overexpression systems for large-scale production of proteins of thermophilic origin. She will be supervising and coordinating Component II that involves cloning, overexpression, and protein purification as well as developing new expression systems for this project.

Component Project Information

The objective of this project is to develop one or more E. coli overexpression systems for Mycoplasma genitalium/M. pneumoniae (from now on referred to as Mycoplasma) genes or their homologs in other organisms, and to purify those gene products selected by Component I for structural studies. A large scale preparation of gene products at greater than 95% purity for structural studies requires two essential steps:

(1) large scale fermentation of cells containing the gene products or overexpression of the recombinant genes in a heterologous host, and (2) large scale purification. Since most of the gene products in Mycoplasma cells or their homologs in other organisms are present at low levels, overexpression of the genes followed by very efficient purification is the only practical process for this project. This component is divided into two parts:

(1) development of new expression systems for Mycoplasma genes or their thermophilic homologs and (2) cloning, overexpression, and purification of the gene products selected according to the criteria listed below.

Four groups of genes for cloning will be selected with preference for thermophilic homologs of Mycoplasma (1) the soluble proteins of Mycoplasma or their thermophilic homologs for which implied cellular functions have been assigned based on sequence similarities but no molecular function or structural information are available; (2) the "hypothetical" Mycoplasma gene products or their thermophilic homologs for which there are neither implied functions nor structural homologs; (3) the Mycoplasma gene products or their thermophilic homologs that belong to sequence families with multiple members; (4) the soluble domains of membrane proteins of Mycoplasma or their thermophilic homologs.