Recombinant Protein Expression revised 8.24.

2005 Scott Lab GLL

Translation issues [4] Translation efficiency can be modulated by changing the distance between the Shine-Delgarno (SD) sequence complementary to the 3 end of the 16S rRNA (consensus 5-UAAGGAGG-3) and the start codon (most commonly AUG and 8% of the time GUG). The composition of the SD sequence can also have an effect on translation efficiency. In some cases, it may be beneficial to decrease translation efficiency if excess protein production is causing formation of inclusion bodies. Also, codon usage can be an issue. See section 2.1.1 of this primer. See also [1, 3].

Secondary structure in mRNA transcript [1, 4] Search coding region for sequences complimentary with ribosome binding site and/or translation start site. Search for other secondary structures which might be present at or around the translation initiation site. Consider replacing codons with equivalent ones to change the sequence and reduce complementarity.

Unexpected stop codons [1] Mutations during PCR amplification of the target gene for cloning can result in creation of stop codons. The final clone should be checked by sequencing to verify that mutations havent occurred. Testing for premature truncation can be done by in vitro translation reactions.

Transcription terminator [1] The presence of a terminator can increase protein expression since the lack of one can result in competing read-through RNAs. This is generally not a problem for pET vectors since most of them have the selective marker gene in the opposite orientation of the target gene. For vectors having the target gene followed by the selective marker gene in the same orientation, check to see if a transcription terminator is present after the target gene. If not, the competition for production of mRNA and protein for both genes may be resulting in decreased levels of your recombinant protein.

Instability of mRNA [1, 3] mRNA of recombinant genes tends to accumulate in the cell; however, E. coli mRNAs are rather unstable. It has been shown that stable secondary structures engineered into the 5 untranslated region and 3 rho-independent terminator of the mRNA can aid in mRNA stability and prevent degradation by exonucleases. In particular, a hairpin at the 5 end without any 5 single-stranded nucleotide overhangs has conferred mRNAs with considerable resistance to exonuclease activity in the cytoplasm.

Insoluble protein Strategies for Soluble Protein Expression Without target modification Decrease culture growth temperature [5]. Growth at 37C can promote inclusion body formation for some proteins while growth at lower temperatures (e.g., 30C, 25C, 15C)

Recombinant Protein Expression revised 8.24.2005 Scott Lab GLL

may not. Growing the culture at a lower temperature will slow the rate of protein synthesis, possibly keeping recombinant proteins from saturating cellular folding machinery and aggregating. The lower temperature also decreases protease activity. Note that growing the culture at a lower temperature will significantly slow the growth of E. coli, and so a longer induction period (e.g. overnight) may be necessary to obtain a sufficient amount of recombinant protein. Change the composition of lysis buffer [1]. A protein may partition into the insoluble fraction of the cell extract because of the composition of the lysis buffer and not because it is located in an inclusion body. Proteins which contain hydrophobic regions, transmembrane domains, or those which associate with membrane-bound proteins may segregate into the insoluble fraction in standard lysis buffers (e.g. with high salt, 0.5 M NaCl). These proteins may be converted to the soluble fraction by addition of millimolar amounts of zwitterionic detergents to the lysis buffer (note that this may affect the purification of the protein later on). Add potential cofactors to the growth medium. Some proteins cannot properly fold without their cofactor and therefore can form inclusion bodies. Do some bioinformatics analysis of the protein sequence to see if it has any homology with proteins known to contain cofactors. This may give you an idea of whether this could be a cause for insolubility for your protein. Alter pH of growth medium [3]. pH is one culture variable that can affect proteolytic activity, secretion and protein production levels. Use other additives during culture growth to promote solubility. See Chapter 3 of [2] for a detailed review. Use different E. coli strains [5]. For codon-usage problems, consider a strain with supplemental tRNA genes. If your protein necessitates disulfide-bond formation for proper folding, consider a strain with enhanced cytoplasmic disulfide-bond formation. Co-express additional chaperones to aid in protein folding. Co-expressing other proteins adds an additional stress on the cells as their expression machinery is more heavily burdened. This can cause a reduction in the expression of your protein, but if it promotes solubility, then it may be worth it. For disulfide bond formation, co-express thioredoxin (or use as a fusion partner) or use strains deficient in thioredoxin reductase. Proteins which require a disulfide bond in order to fold properly or require a disulfide bond for oligomerization may form inclusion bodies if the disulfide bond cannot form. The E. coli cytoplasm is a reducing environment and disulfide bond formation can be difficult unless thioredoxin is present or there is a deficiency in the host thioredoxin reductase responsible for reducing disulfide bonds. An alternative to consider is targeting the protein to the periplasm where disulfide-bond formation can occur (most E. coli proteins having disulfide bonds are located in the periplasm).

With target modification [2-5]. Use fusion proteins as solubility-enhancing partners (e.g. E. coli maltose binding protein (MBP) & N-utilizing substance A (NusA))

Recombinant Protein Expression revised 8.24.2005 Scott Lab GLL

2.2.2 Optimization of Expression You may or may not need to optimize the expression protocol for your protein, and you will need to decide whether this is necessary based on the results of your small-scale test for expression and the type of experiments you plan to do with your purified protein. If you obtained soluble, high-level expression in your small-scale test for expression, you may want to proceed directly to the large-scale expression for protein purification using the same parameters used in your small-scale growth. Alternatively, if you protein expressed poorly or was insoluble, there are some variables you may want to try to optimize to increase expression level or promote solubility. There is no one guaranteed system for optimizing expression, so you will have to devise your own and proceed based on the results you obtain. The guidelines below are meant to help you decide where to start, and the references listed may aid you during the course of optimization. No expression or minimal expression Causes Plasmid instability or plasmid loss [1] (see also Ch 2, 1.1.1 in [2]) pET vectors are usually very stable, but consider the following possibilities. Plasmid loss usually occurs with recombinant toxic genes when antibiotic is not in use since the plasmid-bearing cells will die off due to the toxic gene and leave the remaining cells to overtake the culture. For vectors with selection by ampicillin resistance, the selection can be lost due to degradation of the ampicillin by -lactamase, the ampicillin-resistance gene. -lactamase is produced in abundance and secreted into the media where it degrades the ampicillin, thereby removing the selective pressure for cells to maintain their recombinant plasmids. If you suspect plasmid instability, perform the plasmid stability test outlined in the pET System Manual, Section V. C. Proteolytic degradation of the protein product [1, 3] Protease susceptibility can be affected by the N- and C-terminal sequences of the recombinant protein. The presence of Arg, Leu, Lys, Phe, Trp, or Tyr at the N-terminus targets proteins for more rapid degradation (N-end rule). Keep in mind that N-terminal Met can be removed by E. coli (especially when the amino acid adjacent to the N-terminal Met has a small side-chain), so you should look at the second amino acid from the N-terminus. Non-polar amino acids at the C-terminus can lead to rapid degradation; however, proteins with last five amino acids polar or charged fail to be degraded. Other protease recognition motifs exist; however, they are very diverse and few if any consensus sequences exist. Other factors in protease susceptibility include the presence of damaged or excess protein products caused by formation of incomplete polypeptides, excessive synthesis of subunits from multimeric complexes, post-translational damage, or genetic engineering of the target protein and gulture growth parameters such as nutrient composition of media, growth temperature, and pH. Secondary site translation initiation [1] The presence of a sequence similar to ribosome binding site appropriately spaced (5-13 nt) upstream of an AUG can direct protein synthesis resulting in the formation of truncated protein products.

Recombinant Protein Expression revised 8.24.2005 Scott Lab GLL

Add C-terminal or N-terminal extensions Substitute selected amino acid residues Express protein fragments

Suggested references for trouble-shooting: Makrides, S.C., Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev, 1996. 60(3): p. 512-38. Sorensen, H.P. and K.K. Mortensen, Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact, 2005. 4(1): p. 1. Baneyx, F., Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology, 1999. 10(5): p. 411-421. Balbas, P. and A. Lorence, Recombinant gene expression : reviews and protocols. 2nd ed. Methods in molecular biology ; 267. 2004, Totowa, N.J.: Humana Press. xvi, 506. Villaverde, A. and M.M. Carrio, Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett, 2003. 25(17): p. 1385-95. pET System Manual. Section V. Optimizing Expression. Latest version available online at Novagen / EMD Biosciences website (http://www.emdbiosciences.com/product/TB055). Coligan, J.E. and Wiley InterScience (Online service, http://www3.interscience.wiley.com/cgi-bin/mrwhome/104554813/HOME), Current protocols in protein science, Wiley Interscience: Hoboken, NJ. Coligan, J.E. and Wiley InterScience (Online service, http://www3.interscience.wiley.com/cgi-bin/mrwhome/104554809/HOME), Current protocols in molecular biology, Wiley Interscience: Hoboken, NJ. References 1. 2. 3. 4. 5. (May, 2003). pET System Manual, 10th Edition (Novagen). Balbas, P., and Lorence, A. (2004). Recombinant gene expression : reviews and protocols, 2nd Edition (Totowa, N.J.: Humana Press). Makrides, S.C. (1996). Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60, 512-538. Baneyx, F. (1999). Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology 10, 411-421. Sorensen, H.P., and Mortensen, K.K. (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4, 1.