Question

11. Sometimes the strategy for the expression of a target protein in a host organism involves synthesizing the protein as part of a fusion protein. Why is this approach useful? How is a fusion protein created?

14. A specific target DNA fragment to be integrated into the chromosomal DNA of the host organism can include (1) only the target gene sequence or (2) the entire plasmid, including the target sequence. Explain how each of these results might occur. What are the advantages or disadvantages of the plasmid vector becoming incorporated into the host chromosomal DNA?

Answer 1

11. proteins are widely used as therapeutics such as insulin, antibiotics.These proteins are secretory and current recombinant technolgy research still suffers challenges for efficient expression and downstreaming.Foreign protein most of the time can undergo proteolysis if the protein is expressed in cytosol.So using fusion tags for target protein can improve the yield and solubility of their fusion partners.The mechanism of action can be hypothesized as

a.fusion of a stable or conserved structure to an insoluble recombinant protein may serve to stabilize and promote proper folding of the recombinant protein.

b.Fusion tags may act as a nucleus of folding ‘‘molten globule hypothesis’’.

Advantages of fusion proteins technology

• Catalytic efficiency: Fusion of certain peptides allow for greater catalytic efficiency by altering the tertiary and quaternary structure of the target protein.^[3]
• Solubility: A common challenge in fusion protein design is the issue of insolubility of newly synthesized fusion proteins in the recombinant host, leading to an over-aggregation of the target protein in the cell. Molecular chaperones that are able to aid in protein folding may be added, thereby better segregating hydrophobic and hydrophilic interactions in the solute to increase protein solubility.^[3]
• Thermostability: Singular peptides or protein fragments are typically added to reduce flexibility of either the N or C terminus of the target protein, which reinforces thermostability and stabilizes pH range.^[3]
• Enzyme activity: Fusion that involves the introduction of hydrogen bonds may be used to expand overall enzyme activity.^[3]
• Expression levels: Addition of numerous fusion fragments, such as maltose binding protein (MBP) or small ubiquitin-like molecule (SUMO), serve to enhance enzyme expression and secretion of the target protein.^[3]
• Immobilization: PHA synthase, an enzyme that allows for the immobilization of proteins of interest, is an important fusion tag in industrial research.^[3]
• Crystal quality: Crystal quality can be improved by adding covalent links between proteins, aiding in structure determination techniques

Creation of a fusion protein.

The proteins to be are selected and from the cDNA sequence of the proteins stop codon are eliminated and then the proteins are joined by ligation or overlap extension PCR.That DNA sequence will then be expressed by a cell as a single protein. The protein can be engineered to include the full sequence of both original proteins, or only a portion of either.If the two entities are proteins, often linker (or "spacer") peptides are also added, which make it more likely that the proteins fold independently and behave as expected.

14.

1. In order to incorporate the target sequence directly without vector, the target sequence has to recognized from the DNA sequence and then cleaved using retsriction fragment length polymorphism and then inserted in the medium so that the host accepts it and integrate into its genome.

2.Use plasmid allows expression of gene in the host more accurately.The plasmid carries all the information and neccessary regions for expression of target sequence like promotor regions, enhancers, origin of replication, a selectable marker, and a suitable site for the insertion of a gene such as the multiple cloning site.

Advantages of plasmid vector

• It allows the expression of the gene irrespective the host replication bacause the plasmid has their own replication machinery.They are autonomous.
• Also they allow easy purification of target gene
• there are markers present which help to ensure that deired gene is expressed.

Disadvantage

they cannot accept largeb fragments

Sizes range from 0 – 10kb.

Standard methods of transformation are inefficient.