Recombinant Production of Fibrous Proteins

During the production of a fibrous protein by recombinant methods, the risk of obtaining insoluble aggregates (inclusion bodies) has to be considered. To avoid inclusion body formation, standard described strategies can be applied (19). However, fibrous proteins might present some additional difficulties. Cellular chaperones can participate in the folding and assembly of viral fibers; for example, Hsp70 and Hsp90 are necessary for the formation of the reovirus fibers (20,21). Sometimes specific chaperones are used to ensure correct folding and assembly of fibrous proteins. The T4 short fibers that we describe in Subheading 2.5. use such a specific, phage-encoded chaperone, called gp57. In the absence of this protein, the T4 short fibers form insoluble aggregates when expressed in Escherichia coli; expression in their correctly folded, trimeric state requires coexpression with gp57 (22). gp57 is also necessary for the folding and assembly of long tail fibers, but its mechanism of action is currently unknown. Thus, when a chaperone requirement exists, the fibrous protein of interest has to be overexpressed in the presence of this cellular or virally encoded chaperone.

Generic fiber protein

Generic fiber protein

Virus- Shaft domains Receptor-

binding binding domain domain

Virus- Shaft domains Receptor-

binding binding domain domain

Shortened fiber protein

Fig. 1. Generalized strategy for producing shortened fiber protein fragments suitable for crystallization. Shown at the top is a generic fiber protein with an N-terminal virus-binding domain, central shaft domains, and a C-terminal receptor-binding domain. Asterisks indicate hinge regions. In partially denaturing conditions (which may consist of raising the temperature or adding intermediate concentrations of detergents or denaturants), the protein partially unfolds. Protease is then added, which cleaves off the unfolded domains. Note that although N-terminal unfolding is shown here, unfolding may also take place elsewhere.

Shortened fiber protein

Fig. 1. Generalized strategy for producing shortened fiber protein fragments suitable for crystallization. Shown at the top is a generic fiber protein with an N-terminal virus-binding domain, central shaft domains, and a C-terminal receptor-binding domain. Asterisks indicate hinge regions. In partially denaturing conditions (which may consist of raising the temperature or adding intermediate concentrations of detergents or denaturants), the protein partially unfolds. Protease is then added, which cleaves off the unfolded domains. Note that although N-terminal unfolding is shown here, unfolding may also take place elsewhere.

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