2. A biochemist would like to use E. coli to produce a mammalian protein. The pr
ID: 222222 • Letter: 2
Question
2. A biochemist would like to use E. coli to produce a mammalian protein. The protein is 40 kDa in size with approximately 20% of its mass resulting from polysaccharides. It was found, during previous experimentation, that when isolating the glycoprotein, it was phosphorylated and formed four distinct disulfide bonds. The cloned gene the researcher has been using was prepared through the construction of a cDNA. (a) what type of vector should the researcher use? Describe the vector and identify the sequences and sites required to allow for digestion, ligation, regulation, transcription and translation. (b) Identify a commercially available vector that you would suggest the researcher use and support this suggestion with your reasoning. (You can make tions as needed as long as they are disclosed) Un Slides new englan di hin l a h SExplanation / Answer
Answer:
2. a. E. coli cell lacks the posttranslational modification machinery, so it is difficult to produce the eukaryotic glycoprotein in E. coli with conventional vectors.
Glycosylated recombinant protein has been produced from genetically engineered E. coli with the pgl operon of Campylobacter jejuni. This strain is not commercially available and the glycosylation pattern is different from that of eukaryote, so it is not frequently used.
Recently, researchers successfully engineered an E. coli strain and got N-glycosylation of eukaryotic recombinant protein.
E. coli Shuffle waaL gmd::kan bacterial strain can be transformed with our vector containing the gene of interest to obtain the glycosylated protein with disulfide linkages.
For the cloning experiment, we can use pET28a+ vector with N-terminal his-tag. The restriction enzymes to be used for digestion should not contain these sites within the insert (gene of interest). So, a pair of suitable sites (eg: Bam H1 and Xho1) can be used for cloning.
The protein can be produced through IPTG induction of the transformed bacteria (the E. coli strain mentioned above for glycosylation) in culture.
b. The researcher should use pET28a+ vector for cloning and purification. This strategy produces a cleavable his-tagged protein which can be used to purify the protein in a 2 step process. First step involves affinity purification using a Ni-NTA column and second step involving FPLC.
c. For cloning:
DNA: pET28a+ vector, gene of interest
Restriction enzymes: BamH1, Xho1
Ligation: T4 DNA ligase
Also other reagents such as buffers for PCR, digestion, ligation, etc are required
For purification:
Reagents: Buffers, PMSF, Protease inhibitor cocktail, IPTG, Imidazole, etc.
Enzymes: Thrombin for cleaving his-tag
The is-tag facilitates the purification step through affinity chromatography. His-tag consists of six his residues in succession which binds to the Ni2+ on the Ni-NTA column. Imidazole is used for elution which competes with the his-tag for the Ni2+ sites.
d. The protein so purified from the modified E. coli strain mentioned above would produce glycosylated protiens with disulfide linkages, and would follow the GlcNAc2Man3GlcNAc2 glycosylation pattern.
So, it is expected to be functional to some extent, but it is not expected to be functionally equivalent to the glycoprotein produced in mammalian cells due the more efficient glycosylation pathway in the latter.
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