Production of VNPs, VLPs, and Chimeras - Viral Nanoparticles: Tools for Materials Science and Biomedicine - page 54

..1 E. coli and Other Prokaryoic Expression Systems

E. coli is one of the most widely used hosts for production of heterologous

proteins in general. Its genetics, biochemistry, and molecular biology are
better characterized than any other microorganism. This detailed knowledge
led to the development of sophisticated cloning and expression systems.
Various kits are commercially available that allow manipulation, cloning,
and expression with ease and convenience. The cells have a rapid doubling
time (20-30 min), the growth media are cheap, and E. coli can be produced

in large-scale fermentation systems. These features make E. coli a popular

choice as an expression system.
Several viral coat proteins have been expressed using E. coli , including the

coat proteins of the bacteriophages MS2, Q , HK97, P22, and M13, as well as

the plant viruses CCMV and TMV (Gertsman

b

et al. , 2009; Grieco et al. , 2009;

Hwang et al. , 1994a,b; Kang et al. , 2008; Peabody, 2003; Sidhu, 2001; Strable

et al. , 1995).

A limitation of the

, 2008; Zhao et al.

system is that eukaryotic proteins often
do not assemble correctly, are insoluble, and accumulate in so-called
inclusion bodies. CCMV coat proteins may be expressed to high levels in
E. coli

E. coli

; however, self-assembly into particles could not be achieved (Zhao

et al. , 1995). The produced protein must be further purified, denatured,

refolded, and then assembled into the capsids

(Section 3.5), but
this is cumbersome, inefficient, and not suitable for large-scale production.
To overcome these limitations, alternative expression systems have been
applied. Intact CCMV VLPs were produced and self-assembled using
the prokaryote

in vitro

Pseudomonas fluorescens (Phelps et al. , 2007b). The P.

fluorescens ;

it is easy to manipulate, provides high yields of soluble and active proteins,
and is suitable for large-scale fermentation.
In general, for eukaryotic viruses, it is more common to produce self-
assembled VLPs using a eukaryotic expression system such as yeast or
baculovirus-based expression (Sections 3.2.2 and 3.2.3).

-based expression system is an attractive alternative to E. coli

3.2.1.1 Unnatural amino acid incorporaion into VLPs using a
heterologous expression system

In addition to the creation of mutant particles, expression of VLPs in
heterologous expression system allows the incorporation of unnatural
amino acid side chains into the viral coat proteins. This has been shown