Research Program
Biomolecular Devices and Analysis

Objectives
There are two major goals for this collaborative project: 1) further develop the P-gel system into a robust, efficient, and low-cost protein expression platform, and 2) Use P-gel to produce the CD31 N-terminal domains for crystallization and X-ray structure determination.

Methods
We proposed to develope the P-gel system such that it can be readily used in most labs to produce most functional proteins within 24 hours. In particular, we optimized the reaction conditions and developed the best gel formats (including column, droplets, etc) to further increase the protein yield and efficiency. To this end, we utilized a microfluidic device developed in Luo’s group to produce P-gel in a microdroplet format. This would increase the surface/volume ratio and is expected to enhance the expression yield.  Different cell lysates was also proposed to alter the protein production rate and yield. This is particularly important in reducing the cost of protein production as well as in making sure that the protein can be folded correctly.  One of the key advantages of P-gel is the high surface/volume ratio for fast enzymatic kinetics. In addition to the proposed microdroplets, we are also exploring the nano-fiber format for the P-gel by collaborating with Prof. Harold Craighead, whose group has developed a micro-spinning technology and has successfully spin λ-DNA fibers. We will either incorporate the P-gel gene nano-fiber into the microdropplet, or use the nano-fiber directly in our reaction.

To produce a protein, we “popped” the gene into a P-gel first which can be easily accomplished by digesting the gene with a compatible restriction enzyme (Ava I) before ligating to the X-DNA crosslinkers.

Summary
In summary, we have progressed very well with this project – a P-gel manuscript has been submitted to Nature Materials and now it is in the revision stage.  Another manuscript that is peripherally related has been accepted by Nature Nanotechnoloy. A third manuscript that is also peripherally related is being reviewed by Nature Materials. We are also revising a fourth manuscript for Nature Nanotechnology, this ms is more related to NBTC funded projects.

There is a personnel change during the funded period: Prof. Quan Hao was recruited away from Cornell.  Thus our research plan had to change accordingly.  We have recruited a neuro-biologist from Weill Cornell Medical School , Prof. ChenJian Li, into the project.  Details about this change are elaborated in the following renewal request.

Accomplishments

• Further developed the protein-producing gel system.
• Produced a number of proteins with high yield with the gel (up to 5 mg/ml).
• Started collaboration with Weill Cornell Medical School faculty members