TROUBLESHOOTING BACTERIAL PROTEIN EXPRESSION

Problem	Possible explanation	Solutions
No or low expression	Protein may be toxic before induction	Control basal induction l Add glucose when using expression vectors containing lac-based promoters l Use defined media with glucose as source of carbon l Use playsS/playsE beaning strains in T7-based systems l Use promoters with tighter regulation
	Protein may be toxic after induction	Lower plasmid copy number Control level of induction l Tuneable promoters l Use strains that allow control of induction [Lemo21(DE3)strain] or lacY − strains (Tuner™)
	Codon bias	Lower plasmid copy number Use strains that are better for the expression of toxic proteins(C41 or C43) Direct protein to the periplasm Optimize codon frequency in cDNA to better reflect the codon usage of the host Use codon bias-adjusted strains Increase biomass: l Try new media formulations l Provide good aeration and avoid foaming
Inclusion body formation	Incorrect disulfide bond formation	Direct protein to the periplasm Use E. coli strains with oxidative cytoplasmic environment
	Incorrect folding	Co-express molecular chaperones Supplement media with chemical chaperones and cofactors Remove inducer and add fresh media Lower production rate: l Lower temperature. If possible, use strains with cold-adapted chaperones l Tune inducer concentration

Information from: Recombinant protein expression in Escherichia coli

Inclusion Bodies Formation

The formation of inclusion bodies may proceed through nonpermissive pathways from folding intermediates during the folding process as suggested by Mitraki and King, and temperature is one parameter that affects the conformation and stability of proteins. Increased temperature has been found to stimulate aggregation in several cases. The effect of the induction temperature on the formation of inclusion bodies of SpA-Pgal also indicates that the formation might be caused by hydrophobic interactions between protein chains. A higher temperature increases hydrophobic interaction and might also expose hydrophobic stretches of amino acids that are normally not exposed. The lack of inclusion body formation after changes in the amino acid sequence in the linker region between protein A and 3-galactosidase also indicates that the folding of SpA-Pgal may be important for the formation of a soluble protein. An alternative explanation would be specific intermolecular

interactions of the linker region of SpA-Pgal from pRITL. Thirty-eight of the amino acids in the linker region originate from the C terminal of the lac repressor protein, Lacd. The C terminal of Lacd is not involved in DNA binding but might be involved in binding of inducer or in the formation of its tetrameric structure. The linker region of pRITl-encoded SpA-Pgal is exposed since it is subjected to proteolytic cleavage in the linker by the outer membrane-bound protease OmpT during purification. The linker also contains regions rich in hydrophobic amino acids. Lee and coworkers have shown that when a hydrophobic sequence is introduced between P-galactosidase and a region of the hepatitis B virus surface antigen, inclusion body formation increases with the incubation temperature. The fusion protein without the hydrophobic sequence remained soluble independent of the incubation temperature.

From: Factors Influencing Inclusion Body Formation in the Production of a Fused Protein in Escherichia coli

Bacterial expression system technology platform guarantee protein expression yield and success rate, tech support include:

1. Codon and mRNA structure optimization
2. Vector construction
3. Multiple purification methods
4. Multi-channel protein refolding
5. MALDI-TOF
6. Protein N-terminal sequencing
7. Aliquot and lyophilisation

More information from: http://www.biologicscorp.com/bacterial-system