|
|
 |
|
| L. Jeanne Perry - | |
|
|
Escherichia coli remains the most popular host for heterologous expression
of recombinant proteins. The genetics of this bacterium are the most
characterized and well studied of any microorganism. In addition, E. coli
has many advantages over others hosts including its ease of handling, low
cost, and efficient growth. However, there are several drawbacks that can
hamper high-level expression of recombinant proteins for structural
determination. Among the roadblocks are expression, solubility, folding and
purity.
Currently we are involved in several structural genomics projects:
Pyrobaculum aerophilum, Rhodopseudomonas palustris, Deinococcus radiodurans,
Mycobacterium tuberculosis and others. Pyrobaculum aerophilum is a
hyperthermophilic (Topt = 100C), facultatively aerobic, archeon. Its
sequence contains 2.2 Mb, with 59% G + C (Fitzgibbon et al., 2002). The
structural genomics of Pyrobaculum aerophilum will provide insights for
understanding life in extreme conditions as well as shedding more light on
this understudied branch of life. Mycobacterium tuberculosis, the infective
agent that causes the disease tuberculosis (TB). One-third of the world's
population are infected and about 3 million people die each year from TB.
The genome of Mycobacterium tuberculosis contains 4.4 Mb, with a relatively
high 66% G + C (Cole et al., 1998). Our approach to the Mycobacterium
tuberculosis structural genomics project will yield information for
drug-design and vaccine production against tuberculosis. In addition, we
hope to provide further insights into the mechanisms of mycobacterial
pathogenesis.
The results from these projects, relevant to the bottlenecks of protein
production, as well as recent advances and improvements in expression
technology will be presented. An overview of yeast expression will also be
presented. The basic issues that hamper production of functional protein as
well as strategies to overcome them will be discussed. Despite the
roadblocks, the growing list of solved protein structures have continued to
make Escherichia coli the expression host of choice for the production of
many proteins for structural studies.
|
|
| |
|
|
