Expressing a gene from one organism in the cell of another species has many chal

Question

Expressing a gene from one organism in the cell of another species has many challenges and obstacles, especially since post-translational modification of proteins is so different among organisms. For example, when human recombinant insulin was first expressed in E. coli many strategies had to be devised to achieve proper folding and disulfide bond formation in the insulin. Give two specific examples of a post-translational modifications important is forming a functional protein. For each, suggest a way to recover functionality when making a protein in a non-native organism. Cite your sources.

Explanation / Answer

Protein expression of an organism in another organism is definitely challenging as the microenvironment is different in a non-native organism. This difference in the microenvoronment can lead to various problems like low protein yield, protein toxicity, inclusion body formation,no protein expression, lack of posttranslational modifications, etc.

Both prokaryotic and eukaryotic systems are used for expression of foreign proteins. But both of these systems pose many challenges. Prokaryotic expression systems have higher growth rates and protein prodution may be high. But there are many problems associated with this system like processing of proteins ( no glycosylation, phosphorylation, etc) and improper folding. Protein folding and processing in eukaryotic expression systems may be efficient but the major disadvantage associated with these systems is their slower growth than the prokaryotic systems.

There are many posttranslational modifications necessary for forming a fully function protein.

Two specific examples are

a) Disulphide bond formation

b) Glycosylation (addition of oligosaccharides)

a) First we will discuss about disulphide bond formation in non-native organism. Many proteins have disulphide bonds for attaining their biological activity(e.g insulin). This is carried by Protein Disulphide Isomerase (PDI) In the endoplasmic reticulum in eukaryotoc cells. PDI is also necessary for formation of correct disulphide bonds. In E.Coli PDI is absent. Also the cytoplasm is reducing in nature which reduces disulphide bonds. So proteins with correct disulphide bonds will not be formed.

There are three ways to recover functionality of a protein,

1) to direct the proteins to periplasm which contains Dsb family of enzymes and catalyze disulphide exchange reactions.

2) to use engineered E.Coli strains that have oxidative cytoplasmic environment and favour disulphide bond formation.

3) co-expression of recombinant protein with PDI

b) Now we will discuss about glycosylation.

Glycosylation is important for many proteins to maintain their functionality. Glycosylation may be N-linked or O-linked. N-linked glycosylation confers stability to many secreted glycoproteins. Olisaccharides on glycoproteins may serve as antigens.

Prokaryotes rarely possesess enzymes like oligosaccharyl transferase needed for proper glycosylation of proteins. So proteins with proper glycosylation is not achieved.

One way to recover a properly glycosylated protein in E.Coli is again the co-expression of recombinant protein with enzymes needed for glycosylation of the proten.

(SOURCES:

1) Molecular biology ot the cell by Hansman and Cooper, 6th edition

2) Recombinant protein expression in E.Coli: advances and challenges ( Review article by German L Rosano and Eduardo A Ceccarelli). Frontiers in microbiology)

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