Environmental Engineering Reference
In-Depth Information
4.2.6 S
uBcEllular
t
argEting
/l
ocalization
The choice of subcellular localization of the recombinant protein is dictated by the application. For
in planta
production of cell-wall-modifying enzymes and self-deconstructing biomass, the product
would need to be localized outside of the cell membrane (e.g., in the apoplast). However, subcel-
lular targeting/localization can also affect production levels and product stability as well as ease
of recovery and solubilization; several studies have indicated the effects of subcellular targeting on
heterologous protein production. If the recombinant protein is stable in the apoplast, this provides
significant advantages in terms of recovery and purification because (1) the protein content of the
apoplastic fluid typically corresponds to only a small fraction of the total protein content of the
biomass (<5%), so there are fewer contaminating proteins that must be removed; (2) intracellular
proteases that would be released during extraction/cell disruption are avoided; and (3) low-molecu-
lar-weight intracellular compounds such as phenolics and/or alkaloids that can interfere with protein
recovery are also avoided. Demonstrated initially by Klement in 1965, vacuum infiltration has been
used in many studies to selectively remove extracellular proteins from the intercellular spaces of
plant leaves on a small scale.
Subcellular targeting of proteins to organelles (e.g., chloroplast, vacuole, mitochondria, peroxi-
some) or to the apoplast is typically accomplished through the addition of a transit peptide or secre-
tion signal peptide at the amino or carboxy terminus of the protein, which is then cleaved during the
transport process (Mackenzie 2005). If no secretion signal peptide or transit peptide is included, the
soluble protein remains in the cytosol.
4.2.7 o
ptimization
of
g
EnE
c
onStructS
Because of the degeneracy in the genetic code (multiple codons encode for the same amino acid),
there are many different DNA sequences that can result in the same amino acid sequence. Many
of the target cellulase, hemicellulase, and cell-wall-modifying enzymes come from bacterial or
filamentous fungal hosts that have different preferences in terms of the codons they use for a par-
ticular amino acid and a different GC content than what is found in plants. Because of advances in
speed and reduction in costs, gene constructs for expression of heterologous proteins are now often
chemically synthesized, which has opened up opportunities for optimization of gene constructs that
not only take into consideration codon usage but also other factors such as mRNA structure and
stability. Although there has been significant progress in identifying synthetic gene design rules
for prokaryotic cells such as
Escherichia coli
, design algorithms for reliable, predictable, and high-
level expression of a specific heterologous protein in a particular plant host are not yet available.
However, codon optimization algorithms coupled with synthetic gene design are often used when
expressing a bacterial or fungal protein in plants.
4.3 Plant-made cell-Wall deGradInG enzymes
Table 4.1 lists the endoglucanase, exoglucanases, β-glucosidases, xylanases, and hybrid β-(1-3,1-4)-
glucanases that have been expressed in plants. As can be seen from the table, most of the work has
been done in tobacco, corn, barley, rice, and
Arabidopsis
, generally using stable nuclear transforma-
tion or chloroplast transformation methods. In most cases the enzyme expression level is presented in
terms of percent TSP or units of activity per TSP; however, it is also important to know the enzyme
yield on a gram per kilogram FW or dry weight (DW) tissue basis. For enzymes that contain a cel-
lulose binding domain, it is also useful to assess the “residual” activity associated with the plant
material after homogenization/extraction. Maximum yields are presented in the table, but expression
levels can range over 2-3 orders of magnitude for different independent transformation events.
It is interesting to note that in most of these cases, a significant level of active enzyme can
be produced through judicious choice of the host, subcellular targeting to sequester the enzyme
