P-gel

A High-yield Cell-free Protein Production System

  • Fig. 1: (a) Fabrication of P-gel. X-DNA and gene are crosslinked by DNA ligase to form P-gel. (b) Cell-free protein production with P-gel. P-gel is added to cell lysate containing the components for protein expression. The gene within P-gel is transcribed into mRNA, which is then translated into protein.
  • Fig. 2: (a) Comparison of the Rluc expression yields from P-gel and SPS. (b) Bioluminescence of functional Rluc from P-gel (left) and SPS (right). Fig. 2 has been reprinted with permission from [4].
  • Fig. 3: Time course of functional Rluc expressions of P-gel (open diamond) and SPS (closed square) which are plotted in different Y axis scales (the arrows indicate their corresponding axes). Fig. 3 has been reprinted with permission from [4].
  • From left: Dr. Nokyoung Park, Prof. Dan Luo, Mark R. Hartman, Jason S. Kahn, Edward J. Rice; Cornell University

The investigation of proteins relies on the ability to produce large quantities of functional proteins. Cell-free protein expression systems provide the ability to generate proteins without a living cell, allowing for simplified and diverse applications. The P-gel system discussed here provides a novel cell-free system for producing desired proteins with extremely high yield beyond physiological conditions and biological confinement.

Introduction


Studying proteins and gaining a greater understanding of the role they play in cells not only expands our understanding of how proteins influence cell function, but also opens up the possibility of developing novel protein-based applications. However, in order to effectively investigate these possibilities, high-yield protein production is an essential first step.

Traditionally, animals or cell cultures are used for protein production. However, these procedures are generally labor intensive, costly, and time consuming. In addition, some proteins can be very difficult to produce and purify. Advancements in the field have lead to cell-free expression where a gene template, in suspension, is used to produce a protein without the requirement of any living cells. Cell-free expression utilizes a purified cell extract (lysate) that is derived from sources like wheat germ, E. coli, or rabbit reticulocyte to transcribe and translate, allowing for faster and simplified protein production [1-3].
In this article, we discuss a novel, gel-based cell-free expression system. This system incorporates a gene into the matrix of a protein producing gel (P-gel) composed entirely of DNA. This is the first time that a gel is used for cell-free expression, providing an alternative approach and higher yield compared to current systems [4].

P-gel: A Gel Made Entirely from DNA

The ability to produce DNA structures is well known, and these molecules can be tailored to fit applications ranging from sensing to electronics [5-7]. In the case of P-gel, the high yield of protein arises from its unique format, composed of a gene sequence and rationally-designed X-shaped DNA (X-DNA) [8-10].

Through unique overhang sequences, each of the X-DNA contains sticky ends that are complementary to those flanking the gene. Such a design allows for crosslinking via ligation of X-DNAs and the genes (fig. 1).

How exactly does P-gel increase the protein yield? There are at least three mechanisms that are responsible for the high yield [4]. First, by capping the gene template with X-DNA, genes are protected from degradation and denaturing, leading to increased stability and prolonged expression. Second, by allowing multiple copies of a gene to link to the X-DNA, higher local gene concentrations are present in the P-gel than would be in a traditional cell-free approach where the gene is in a solution-phase system (SPS). Third, by confining the gene templates through crosslinking, the enzymatic turnover rate increases and facilitates higher rates of protein production.

Protein Production from P-gel

To demonstrate protein expression from the P-gel system, we produced the reporter protein Renilla luciferase (Rluc) with the P-gel using the same conditions as those for the SPS. As shown in figure 2, the P-gel exhibited a ~100-fold enhancement in both yield and efficiency over the SPS control.
By altering the established conditions of SPS, we further improved P-gel protein yield. For example, by extending the reaction time to 36 hours, the P-gel continued to produce proteins resulting in an approximately 260% increase in yield compared to P-gel with a 12-hour reaction (fig. 3). Similarly, by doubling the amino acid amount, P-gel showed a 52% increase. In addition, by refreshing the feeding buffer twice, P-gel achieved a further increase in Rluc production of up to a 270% increase in yield. Using these optimized conditions for P-gel, the overall yield of functional Rluc reached 5.0 mg/ml, representing a 288-fold higher efficiency than SPS. Furthermore, P-gels can be reused up to 5 times without a significant loss in productivity.

Importantly, we demonstrated the potential of the P-gel system as a universal protein production system. We tested the P-gel system with 16 different proteins, including reporter proteins, membrane proteins, kinases, and toxic proteins with molecular weights ranging from 16 kDa to 110 kDa. All 16 proteins were successfully produced from the P-gel system with total protein yields of approximately 1 to 7 mg/ml.

Conclusion

By crosslinking the gene within the scaffolding of P-gel, we created a new gene format and thus dramatically increased the efficiency and yield of cell-free protein production compared to SPS. P-gel is compatible with all lysates and current cell-free protein production methodologies and is a platform technology.

Uniquely, the gel-phase format of P-gel enables applications that cannot be achieved with conventional solution-phase formats. For instance, P-gel has potential applications in biomaterials, tissue engineering, and drug delivery. The inclusion of multiple genes within the P-gel could allow for the construction of metabolic networks, offering possibilities with synthetic biology. P-gel is also compatible with microfluidic or column-based continuous-flow platforms. Coupled with its dramatically high yield, the P-gel system can lead to large-scale parallel and automated protein production.

References
[1] Spirin A.S. et al.: Science 242, 1162-1164 (1988)
[2] Spirin A.S. and Swartz J. R. (ed.): Cell-free protein synthesis, Wiley-VCH Verlag, Weinheim, 2008
[3] Kim D.M. and Swartz J.R.: Biotech. Bioeng. 66, 180-188 (1999)
[4] Park N. et al.: Nature Mater 8, 432-437 (2009)
[5] Seeman N.C.: Nature 421, 427-431 (2003)
[6] Feldkamp U. and Niemeyer C.M.: Angewandte Chemie-International Edition 45, 1856-1876 (2006)
[7] Feldkamp U. et al.: Angewandte Chemie International Edition 48, 2-7 (2009)
[8] Li Y.G. et al.: Nature Mater. 3, 38-42 (2004)
[9] Um S.H. et al.: Nature Mater. 5, 797-801 (2006)
[10] Um S.H. et al.: Nature Protoc. 1, 995-1000 (2006)

 

Authors:
Dr. Dan Luo, Associate Professor
Dr. Nokyoung Park, Research Associate
Mark R. Hartman, Graduate Student
Jason S. Kahn, Graduate Student
Edward J. Rice, Technician

Dept. of Biological & Environmental Engineering, Cornell University

 

Contact

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Ithaca, NY 14853
USA
Phone: +1 607 255 8193
Telefax: +1 607 255 4080

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