1. Three major factors influence protein expression Host Growth Conditions Vector Thus, you should consider the solutions for YOUR expression problems at the levels of vector, strains and induction conditions.

2. No / Low Protein Production Growth ConditionsHost StrainVectorReason Start induction at higher OD Shorten induction time Add Glucose to suppress leaky expression Use BL21AI or BL21(DE3)pLysS/E Use T7 promoter-based vectors (also arabinose) Tightly regulate induction with lac operator Toxic protein Re-clone with more A residues at 5’ Shorten distance between RBS and first ATG (2-8 nt( Initiation problems Slow translation by reducing temperature or grow in poor media Use stains supplementing rare codons (Rosetta, Codon +) Mutate gene for codon optimization Rare codons Start from freshly transformed bacteria Add Glucose to suppress leaky expression Use recA- strains (HMS174; BLR) Tightly suppress gene expression prior to induction Use low-copy origin of replication plasmid Your gene induces rearrangement and loss of the DE3 lysogen Use RNAse deficient strain (BL21Star) Change vector to structured RNA vector RNA degradation

3. Growth ConditionsHost StrainVectorReason Lowaring induction temperature usually helps Use Trx(-)/gor(-) strains (e.g. Origami) for creating oxidative conditions in cytosol Use thioredoxin, DsbA, DsbC fusion partners Clone in a vector containing secretion signal to the periplasm (pelB, OmpA) Protein is misfolded due to lack of correct disulfide bond formation Slow expression rate (low temp; low [inducer]; short induction time; poor media) Heat shock with chemical chaperones Membrane rich strains (C41/C43) Solubility enhancing fusion proteins (MBP, NusA, GST, etc.) Hydrophobic protein Heat shock with chemical chaperones Screen various expressing strains Add vectors for various chaperone co-expression No appropriate chaperones Induce at low temp.Membrane rich (C41/C43) Replace with bacterial signals (secretion) or omit signals Sub-cellular localization signals Membrane rich (C41/C43) Use mistic fusion protein. Generate truncated forms of protein (soluble domains) Membrane proteins Heat shock with chemical chaperones Transform with a partner: combination of 2-4 vectors for max 8 proteins Protein is part of a complex Aggregation

4. Truncated protein Growth ConditionsHost StrainVectorReason Slow elongation by low temp.; low inducer; poor media Use rare codon strains (rosetta, codonPlus) Optimize codon usage Rare codons Slow expression rate with low temp.; low inducer; short harvest; poor media Sub-clone with another fusion partner or avoid N- terminus fusion protein Faster, uncoordinated- translation of fusion protein Grow and induce at low temp, use protease inhibitors when breaking the cells on ice Induce at higher OD and reduce induction time Low protease strains (BL21 derivatives, M15) Detect and replace specific protease sites Degradation