3. Translational fusion Assume the restriction site identified in the gene which you want to express is a BamHI site. Digest with BamHI to obtain: GATCCXXXXXXXXXXXX GYYYYYYYYYYYYY ↓ Treat with Klenow fragment to fill in the unpaired bases to obtain: GATCCXXXXXXXXXXXX CTAGYYYYYYYYYYYYY ↓ Determine the proper reading frame of the gene. Assume the coding sequence of the filled-in fragment should read: GA TCC XXX XXX XXX ↓ Determine which restriction endonuclease should be used to digest an expression vector pSKF301 in order to allow expression of the fusion protein. For this example, StuI is required to yield: ccatg gat cat atg tta aca gat atc aag gGA TCC XXX XXX pSKF301 (carrier sequence) your fusion gene

4. Where to express the recombinant proteins? 1.Direct expression (cytosol): E. coli cytoplasm is a reducing environment - difficult to ensure proper disulphide bonds formation. 2. Fusion expression (inclusion body?): Ensures good translation initiation. Can overcome insolubility and/or instability problems with small peptides. Has purification advantages based on affinity chromatography. 3. Secretion (periplasm or medium): a fusion alternative when proteins are fused to peptides or proteins targeted for secretion. Periplasm offers a more oxidizing environment, where proteins tend to fold better. Major drawbacks: limited capacity for secretion (0.1-0.2% total cell protein compared to 10% produced intracellularly) and inability for posttranslational modifications of proteins.

5. General problems with heterologus gene expression (a) Not enough protein is produced: * codon usage preferential (rare codon) * potential mRNA secondary structure.(5’-end ATcontent, 3’-end transcriptional terminator) * toxic gene. (b) Enough protein is produced, but it is insoluble: * vary the growth temperature. * change fermentation medium. * low-copy-number plasmas. * selection of promoter. The KEY idea is to slow down the expression rate of protein.

6. Insolubility of heterologous proteins produced in E.coli Inclusion bodies. Dense particles, containing precipitated proteins. Their formation depends on protein synthesis rate, growth conditions. Advantages: proteolysis resistant, big yield, relatively pure, easy to separate. Disadvantages: inactive product requires in vitro refolding and renaturation

8. Refolding of recombinant proteins Solubilisation: High T 0 C, detergents, high concentration of inorganic salts or organic solvents all used. The most commonly used organic solutes such as urea or guanidine-HCl often used in the presence of reducing agents (mercaptoethanol or DTT). Solubilized proteins can be purified by ion- exchange chromatography or other conventional methods, prior to refolding. Refolding: If no S-S bonds present - remove denaturing agent to allow protein to fold correctly. If S-S bonds present - their formation can be accomplished: by air oxidation, catalysed by trace metal ions; by a mixture of reduced and oxidized thiol compounds - oxidized DTT, reduced DTT; GSSG/GSH; cystine and cysteine, cystamine and cysteamine.

9. 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

10. 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

11. 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