小中大
FMDV的3C蛋白酶表达，文献上已经有人做过了。 cuturl('http://scripts.iucr.org/cgi-bin/paper?en5101')
cuturl('http://jvi.asm.org/content/81/1/115.full?view=long&pmid=17065215')
cuturl('http://upetd.up.ac.za/thesis/available/etd-08042008-104418/')
增强蛋白质可溶性表达的策略：
Improving Protein Solubility
In many cases the expressed protein is insoluble and accumulates in so-called inclusion bodies. This is especially true under conditions of high level expression. Several strategies are available to improve the solubility of the expressed protein.
Reducing the rate of protein synthesis.
This can be done by:
lowering the growth temperature. This decreases the rate of protein synthesis and usualy more soluble protein is obtained.
using a weaker promoter (e.g. trc instead of T7).
using a lower copy number plasmid.
lowering the inducer concentration.
Changing the growth medium:
addition of prostethic groups or co-factors which are essential for proper folding or for protein stability.
addition of buffer to control pH fluctuation in the medium during growth.
addition of 1% glucose to repress induction of the lac promoter by lactose, which is present in most rich media (such as LB, 2xYT).
addition of polyols (e.g. sorbitol) and sucrose. The increase in osmotic pressure caused by these additions leads to the accumulation of osmoprotectants in the cell, which stabilize the native protein structure.
addition of ethanol, low molecular weight thiols and disulfides, and NaCl.
(Georgiou, G. & Valax, P. (1996) Current Opinion Biotechnol. 7, 190-197)
Co-expression of chaperones and/or foldases.
Two classes of proteins play an important role in in vivo protein folding.
Molecular chaperones promote the proper isomerization and cellular targeting by transiently interacting with folding intermediates. The best characterized E. coli systems are:
GroES-GroEL
DnaK-DnaJ-GrpE
ClpB
Foldases accelerate rate-limiting steps along the folding pathway. Three types of foldases play an important role:
peptidyl prolyl cis/trans isomerases (PPI's)
disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC)
protein disulfide isomerase (PDI) - an eukaryotic protein that catalyzes both protein cysteine oxidation and disulfide bond isomerization. It also exhibits chaperone activity.
Co-expression of one or more of these proteins with the target protein could lead to higher levels of soluble protein. The levels of co-expression of the different chaperones/foldases have to be optimized for each individual case. DsbA and DsbC have also shown possitive effects on expression levels when used as a fusion partner.
Periplasmic expression:
Secretion of the target protein to the periplasm has a number of distinct advantages:
the oxidizing environment of the periplasm allows for the formation of disulfide bonds, which does not occur in the reducing environment of the cytoplasm.
the periplasm contains two foldases, disulfide oxidoreductase (DsbA) and disulfide isomerase (DsbC), that catalyze the formation and isomerization of disulfide bonds.
reduced proteolysis (since less proteins are present).
allows for the accumulation of proteins that are toxic in the cytoplasm.
engineering of an authentic N-terminus.
