Difference between revisions of "Part:BBa K3187017"

Profile

Usage and Biology

Coat Protein is an umbrella term for many different proteins, which simplify the transfer of molecules between different compartments that are surrounded by a membrane. ^[1] We focused on the viral and bacteriophagic coat proteins, which are parts of their respective organisms’ capsid. The genetic information (DNA or RNA) is wrapped and protected by the capsid. During cell infection, the phage or virus transfer the genetic information into the infected cell. ^[2] Because of the high variety of coat proteins, we are focussing on one specific coat protein (BBa_K3187017), which is naturally found in the bacteriophage P22. This coat protein (CP) consists of 431 amino acids and its molecular weight is 46.9 kDa. Because of its significance as a part of the capsid, it represents one main part of our Virus-like particle. Together with the scaffold protein (BBa_K3187021), they assemble to a VLP ^[3] and form the basis for our modular platform.

Methods

The basic part coat protein is produced and purified as the composite part (BBa_K3187000) (CP-LPETGG) and (BBa_K3187001) (CP). For gene expression and protein purification as CP-LPETGG the coding sequence contains a coat protein (BBa_K3187017) a LPETGG (BBa_K3187019), a T7 promoter, lac-operator and RBS (BBa_K3187029), a Short Linker 5AA (BBa_K3187030) T7 terminator (BBa_K3187032), and Strep-tagII (BBa_K3187025). The composite part coat protein without LPETGG (BBa_K3187001) contains a T7 promoter, lac-operator and RBS (BBa_K3187029), two terminators (T7Te terminator and rrnb T1 terminator (BBa_K3187036)), a Short Linker 5AA (BBa_K3187030) and Strep-tagII(BBa_K3187025). The production is performed in the E. coli strain BL21 (DE3) and it is purified with GE Healthcare ÄTKA Pure machine which is a machine for FPLC. To verify the successful production, a western blot is carried out.

Results

The basic part coat portein was expressed, produced and purified as the composite part BBa_K3187000 (coat protein with LPETGG) BBa_K3187001 (coat protein). The production is performed in E. coli BL21 and it is purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. To verify the right production, a western blot is made.

Figure 1: western blot of all produced and purified proteins.

Fig. 1 shows that the band of the CP-LPETGG is approximately found by the 49 kDa band and the band of CP by 46.9 kDa. Consequently, the successful production was proven. CP-LPETGG and CP were detected with Strep-Tactin-HRP.

References

1. ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [1]
2. ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, 80- 88 [2]
3. ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [3]