Part:BBa_K3187001
P22 Bacteriophage Coat Protein expression cassette
Coat Protein
| name | coat protein |
| base pairs | 1293 |
| molecular weight | 46,9 kDa |
| origin | bacteriophage P22 |
| parts | coat protein, T7Te promoter, lac-operator, rrnb T1 terminator, Strep-tagII |
| properties | Assembly with scaffold protein to a Virus-like particle |
Usage and Biology
This part encodes the coat protein (CP) (BBa_K3187017)
of the bacteriophage P22 capsid. Importantly, it must not be confused with coat
proteins in membrane transport of eukaryotic cells. 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]
In the natural context of P22, its genetic information is included
and protected by the capsid, before it is transferred into the host organism during infection.
Bacteriophagic coat proteins have been used for many purposes, for example vaccines or drug delivery.
[2]
The P22 coat protein consists of 431 amino acids
and its molecular weight is 46,9 kDa.
Because it is found in the structural components of viral proteins, it is an important part of Virus-like particles
(VLP) as well. Together with the scaffold protein (BBa_K3187021), the proteins assemble to a VLP and build the basis for our
modular platform.
The coat proteins (BBa_K3187001) are heterologously expressed in E. coli BL21 (DE3). As backbone the pACYCT2 plasmid is used, containing a T7 promoter, lac-operator and RBS (BBa_K3187029). Moreover the part comprises a C-terminal StrepTagII (BBa_K3187025), a Short Linker (5AA) (BBa_K3187030) and two terminators, T7Te terminator and rrnb T1 terminator (BBa_K3187036).
Methods
Cloning
The sequence of the coat protein ordered from Integrated DNA Technologies (IDT) was inserted in the pACYCT2 backbone. For this purpose, the Gibson asssembly was used. The sequence was verified by sanger sequencing through Microsynth Seqlab.
purification
The heterologous expressed coat protein in E. coli was purified using a GE Healthcare ÄKTA Pure machine which is a machine for FPLC
SDS-Page and Western blot
To verify that the coat protein was heterologous produced, a SDS-Page followed by a Western blot was performed.
Results
Cloning and Expression
The ordered sequence from IDT was cloned into the pACYCT2 plasmid with Gibson assembly and heterologous expressed in E. coli. The accuracy of cloning was controlled via sanger sequencing (Microsynth Seqlab ) and the expression was observed using an SDS-Page and Western blot. The Western blot showed a band corresponding to the size of approximately 46,9 kDa. So, the successful cloning and expression was proven.
Figure 1: Western blot of all produced and purified proteins.
Fig. 1 shows that the CPs were detected with anti-strep antibody. The band of the CP is approximatley found by the 46.9 kDa band. Consequently, the successful cloning and expression was proven.
References
- ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [1]
- ↑ Rohovie, Marcus J., Maya Nagasawa, and James R. Swartz. "Virus‐like particles: Next‐generation nanoparticles for targeted therapeutic delivery." Bioengineering & translational medicine 2.1 (2017): 43-57 [2]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1458
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1504
| None |