Clean Genome E.coli – Improved Production

Less is Better - Scarab Genomics bioengineered Clean Genome® E. coli by removing over 15% of the K-12 genome, providing enhanced genetic stability, final products free from insertion sequences (IS), and easier downstream purification away from bacterial components, including endotoxin.

Biopharmaceutical production
The rapidly increasing importance of plasmid DNA, antibodies, and other proteins as biopharmaceuticals has generated a growing demand for economical methods of producing purer, safer recombinant products. Using E. coli as a production host provides one of the fastest, least expensive, and highest product-to-volume methods available for biopharmaceutical production. Some of the shortcomings of using E. coli for production include potential instability or modification of plasmid DNA, endotoxin or other contaminants in final products, and the inability to solubly express some complex proteins. Scarab Genomics’ Multiple Deletion Series (MDS™) E. coli eliminate or reduce these problems, expanding the use of E. coli as a production method for recombinant DNA and proteins.

Clean Genome® E. coli
Using rational design and synthetic biology methods, the K-12 genome was reduced by making a series of precise deletions to remove known recombinogenic and mobile DNA elements, cryptic, virulent genes, and non-essential genes, while maintaining robust growth and protein production (Kolisnychenko). Genome reduction optimizes Clean Genome E. coli strains as biological factories, providing enhanced genetic stability, final products free from insertion sequence (IS) elements, and easier downstream purification away from bacterial cellular components, including endotoxin. Clean Genome E. coli provide ideal strains for a wide spectrum of applications ranging from routine cloning to biopharmaceutical production.

Recombinant DNA
Plasmid DNA as a biotherapeutic is being used for DNA vaccines, RNAi, and gene therapy. One of the great advantages of DNA-based therapies over therapeutic proteins or small molecules is the ability to quickly and exactly design the specific DNA sequence required (Carnes). However, as with most new technologies, a number of design, production, and quality problems need to be addressed. Specifically, low production yields, insertion of transposable elements into the plasmid during production, and maintaining DNA stability and supercoiling in the presence of palindromic and other problematic sequences are important concerns (Prather). Scarab’s strains are designed to improve production levels, eliminate transposable elements, and maintain DNA stability.

IS elements
Contamination of cloned DNA with insertion sequences is an extremely prevalent, but frequently overlooked, phenomenon. To demonstrate the problem of IS contamination, pBR322, purchased or purified from Scarab’s MDS42 E.coli, was used as template in PCR reactions with primer sets designed to amplify IS1, IS2, IS3, IS5, IS10 and IS186 insertion sequences. Two sets of primers were used for each IS element: an outward, back-to-back set to amplify circular (i.e., plasmid) DNA and an inward set to amplify linear (i.e., genomic) DNA. Unexpected, small PCR products generated with outward primers were cloned and sequenced, revealing that the small products were mini-circles formed by free insertion sequences that were co-purified with full length plasmids. Additional experiments comparing the presence of IS elements in Clean Genome strains with commonly used E. coli strains have been published (Posfai). It should be noted that IS contamination is so prevalent that significant plasmid screening was necessary to find a starting plasmid without IS elements to transform MDS42 for these experiments.

Inward primers (A-D) or outward primers (E-H) specific for IS1, IS2, IS3, IS5, IS10 and
IS186 were used for PCR (lanes 1-6, respectively; M, 1 kb-plus size standard). (A, E) negative
controls (no DNA); (B, F) positive controls are the individual IS elements cloned into
pBR322. (C, G) purchased pBR322; (D, H) pBR322 isolated from MDS42 (Posfai).
From Science.  Reprinted with permission from AAAS. Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes.  Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher.

DNA stability
Typically, lentiviral expression clones containing long terminal repeats (LTRs) are unstable in standard E. coli cloning hosts, making the viral DNA difficult to clone. Frequently regions between the LTRs are deleted, which is believed to be due to homologous recombination (Das Gupta). Even hosts specifically designed for cloning lentiviral LTRs have proven to be less than optimal. Chakiath and Esposito (2007) showed that MDS42 maintained stable lentiviral expression clones containing direct repeats, and outperformed both common and specialized cloning strains.

Adeno-associated viral (AAV) vectors contain inverted terminal repeat sequences (ITRs), which fold to form stable secondary structures that are also typically deleted in E. coli. To compare the stability of ITRs in MDS42 and its parent strain, MG1655, the strains were transformed with plasmid pT-ITR, grown over serial subcultures, and assayed by restriction enzyme digest to look for DNA patterns. DNA produced in MDS42 gave uniform digest patterns over 4 serial subcultures; DNA produced in MG1655 gave multiple new DNA fragment sizes. Sequencing confirmed that DNA produced in MG1655 lost ITRs.

The AAV ITR (viral ends) folding to form a hammerhead is shown above the gel photo. Stability of pT-ITR was monitored by restriction enzyme digests with KpnI (a), MscI (b), and NotI  (c)  assayed by agarose gel electrophoresis.  Lanes 0, primary cultures from freshly transformed bacteria; lanes 1-4, subcultures 1-4. Fragment sizes in lanes 0 are consistent with predictions from the pT-ITR sequence.  KpnI linearizes the plasmid; accumulation of a smaller linear fragment appears for DNA produced in MG1655. NotI cuts outside each of the hammerheads to release a 1.6 kb fragment, which is gradually lost in MG1655 DNA. MscI cuts at the 5′ end of the stem to release 1.2 kb fragment from between the hammerheads. For DNA produced in MG1655 progressive changes are seen that are consistent with loss of the hammerhead region (Posfai). From Science.  Reprinted with permission from AAAS. Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes.  Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher.

Recombinant proteins
Therapeutic proteins offer potential advantages over traditional drug therapies because they more specifically target the cause of the disease, rather than just the symptoms, resulting in higher efficacy and fewer side effects. For simple proteins, E. coli is the preferred production organism because it offers high productivity at a relatively low cost; this organism grows quickly on inexpensive media and can accumulate large amounts of the recombinant protein, up to a substantial proportion of total cell protein. Productivity in E. coli can result in over 10 grams of protein per liter of culture within 20-30 hours. Proteins with multiple chains, multiple disulfide-bonds, or requiring extensive post-translational modification have been more difficult to produce in E. coli using current hosts, so they are made in insect or mammalian cell lines.

Compared to E. coli, insect and mammalian cell lines are slower growing, less productive, and more expensive. Using mammalian cells causes greater safety concerns, principally due to the risk of contaminating the therapeutic product with dangerous endogenous and adventitious agents, such as mammalian viruses. In spite of disadvantages, the increasing demand for therapeutic proteins has created a serious global shortage of mammalian cell culture capacity.

Improved protein yields
Although current E. coli production expression strains are deficient for productive phage, they still contain prophage elements that can retain lysis and recombination functions. Under the stress of over-expression and fermentor growth conditions, prophage can be activated and cause premature lysis and lower yields. For additional information and data on premature lysis during T7 protein expression in a Clean Genome Strain compared to BL21(DE3), please see: Scarab Genomics Product Info (Pgs 5-6)

Scarab’s Clean Genome strains offer increased productivity using rich or minimal medium. Evidence suggests that these strains will also significantly expand the repertoire of proteins that can be made in E. coli. In addition, the reduced E. coli genome can simplify purification procedures, resulting in cost savings and purer, safer therapeutic proteins.

About Scarab Genomics
In 1997 Scarab Genomics founder Fred Blattner, working at the University of Wisconsin - Madison, published the first complete genomic DNA sequence of an E. coli, strain K-12, which is widely used in recombinant DNA research and for production of therapeutics such as insulin. Although K-12 is generally regarded as safe and is exempt from the Recombinant DNA Guidelines of the NIH, Blattner and his team noted that the K-12 genome contained many genes that, if removed, could provide a greater level of safety, increase production efficiency, and improved genomic and plasmid DNA stability. Scarab Genomics, LLC was organized in February 2002 to develop and commercialize the core technical competencies in genome engineering and bacterial functional genomics. The company designs genetic modifications in bacteria to achieve a particular objective and can predict, with reasonable accuracy, the likely outcome. Since genome design is not an exact engineering science, Scarab combines this predictive approach with a complementary empirical approach, in which the company’s expertise to recognize, investigate, and understand secondary effects on cellular pathways and function is vital.

Conclusion
When at all possible, E. coli has been the preferred production host for recombinant DNA and protein manufacturing. Clean Genome strains offer many unique advantages over traditional, commonly used E. coli hosts, overcoming previous technological limitations of this host and enabling E. coli to be used for:
• A wider range of products
• Increased productivity
• Reduced production costs
• More reproducible and controllable production processes
• Simpler downstream processing from less complex starting material
• Improved purity and safety of the final product

MDS strains with additional genome deletions have been made and are currently being tested.

References
• Carnes, A.E. and Williams, J.A. 2007. Plasmid DNA manufacturing technology. Recent. Patents on Biotechnology. 1(2): 1-16.
• Chakiath, C.S. and Esposito, D., 2007. Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43, 466-470.
• DasGupta, U., Weston-Hafer, K., Berg, D., 1987. Local DNA sequence control of deletion formation in Escherichia coli plasmid pBR322. Genetics 115, 41-49.
• Kolisnychenko V., Plunkett G. 3rd, Herring C.D., Feher T., Posfai J., Blattner F.R., Posfai G. 2002. Engineering a reduced Escherichia coli genome. Genome Res. 12(4): 640-7.
• Posfai, G. et al., 2006. Emergent properties of reduced-genome Escherichia coli. Science (supporting online material) 312, 1044-1046.
• Prather, K.L.J., Edmonds, M.C., Herod, J.W. 2006. Identification and characterization of IS1 transposition in plasmid amplification mutants of E. coli clones. Appl. Microbiol Biotechnol. 73:815-826.