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Three major factors influence protein expression

Vector

Host

Growth Conditions
Thus, you should consider the solutions for YOUR expression problems at the levels of vector, strains and induction conditions.

No / Low Protein Production


Reason Toxic protein Vector Use T7 promoter-based vectors (also arabinose) Tightly regulate induction with lac operator Re-clone with more A residues at 5 Shorten distance between RBS and first ATG (2-8 nt( Host Strain Use BL21AI or BL21(DE3)pLysS/E Growth Conditions Start induction at higher OD Shorten induction time Add Glucose to suppress leaky expression

Initiation problems

Rare codons

Mutate gene for codon optimization

Use stains supplementing rare codons (Rosetta, Codon +)


Use recA- strains (HMS174; BLR)

Slow translation by reducing temperature or grow in poor media


Start from freshly transformed bacteria Add Glucose to suppress leaky expression

Your gene induces rearrangement and loss of the DE3 lysogen RNA degradation

Tightly suppress gene expression prior to induction Use low-copy origin of replication plasmid Change vector to structured RNA vector

Use RNAse deficient strain (BL21Star)

Aggregation
Reason Protein is misfolded due to lack of correct disulfide bond formation Hydrophobic protein Vector Use thioredoxin, DsbA, DsbC fusion partners Clone in a vector containing secretion signal to the periplasm (pelB, OmpA) Solubility enhancing fusion proteins (MBP, NusA, GST, etc.) Host Strain Use Trx(-)/gor(-) strains (e.g. Origami) for creating oxidative conditions in cytosol Membrane rich strains (C41/C43) Growth Conditions Lowaring induction temperature usually helps

Slow expression rate (low temp; low [inducer]; short induction time; poor media) Heat shock with chemical chaperones Heat shock with chemical chaperones

No appropriate chaperones

Add vectors for various chaperone co-expression

Screen various expressing strains

Sub-cellular localization signals


Membrane proteins

Replace with bacterial signals (secretion) or omit signals


Use mistic fusion protein. Generate truncated forms of protein (soluble domains) Transform with a partner : combination of 2-4 vectors for max 8 proteins

Membrane rich (C41/C43)


Membrane rich (C41/C43)

Induce at low temp.

Protein is part of a complex

Heat shock with chemical chaperones

Truncated protein
Reason Rare codons Vector Optimize codon usage Host Strain Use rare codon strains (rosetta , codonPlus) Growth Conditions Slow elongation by low temp.; low inducer; poor media Slow expression rate with low temp.; low inducer; short harvest; poor media Low protease strains (BL21 derivatives, M15) Grow and induce at low temp, use protease inhibitors when breaking the cells on ice Induce at higher OD and reduce induction time

Faster, uncoordinatedtranslation of fusion protein Degradation

Sub-clone with another fusion partner or avoid N-terminus fusion protein Detect and replace specific protease sites