Question

After establishing the protein expression laboratory for your company, you have been asked to produce human insulin protein in bacteria. Although you do not have to worry about purification (that is another lab), you do have to express the protein at sufficiently high levels. Note that the gene has already been placed behind a bacterial promoter to allow for bacterial transcription. Answer the following questions regarding this expression:

a) What component or components of the insulin gene will need to be changed in order to get proper expression in bacteria? Why?

b) What would happen if the 5’ cap of the RNA could not be added? Why?  

c) What would happen if GTPase (an enzyme that converts GTP to GDP) activity was blocked? Why?

Answer 1

a. Post translational modifications such as glycosylation, phosphorylation, proteolytic processing and formations of disulfide bonds do not occur in bacteria as they are only seen in eukaryotic cells.

Insulin is produced by beta cells of islets of Langerhans in the pancreas. It is produced as a pre-proinsulin molecule with an N terminal signal peptide that causes its entry into ER. Cleavage of signal peptide in ER lumen leads to formation of the proinsulin. In the ER, three disulphide bonds are formed to form folded proinsulin, which then enters the trans-Golgi network. Prohormone enzymes (PC1 and PC2) and exopeptidase carboxypeptidase E actions will lead to formation of active insulin.

Although the bacterial promoter is used for production of insulin in bacteria, eukaryotic insulin genes have introns, which cannot be cannot be cleaved by bacteria due to lack of alternative splicing. Hence, mRNA for eukaryotic insulin is first converted to CDNA which is then inserted in expression vector linked to bacterial promoter. Ability to add a 5’ cap is also a required as bacterial system lack 5’ cap on mRNA. Further, there should be poly A tail present.

Insulin can be synthesized in E.coli as two chains A and B. These chains are then joined under optimal conditions to produce active insulin. An alternative approach is to synthesize the single polypeptide, purify it and then excise the C-peptide by proteolytic digestion. Campylobacter jejuni is able to glycosylate proteins by N-glycosylation. This system of C. jejuni can be transferee to E.coli in order to glycosylate insulin. Pglb oligosaccharyltransferase from C. jejune has been tried to be expressed in E.coli, although this glycosylation is slightly different than in eukaryotes. In order to increase the solubility of the protein, chaperones can be added to bacterial system to cause proper folding of the protein.

Amino acid residues B8, 9, 12, 13, 16 and 23-28 in the B chain of insulin are involved in dimer or oligomer formation in insulin. These amino acids can be modified in E.coli insulin to make it a fats acting insulin by increasing higher concentrations in blood.

b. A 5’ cap (5’ m7G) is added to mRNA by guanyl transferase between the 5’ end of the mRNA and GTP molecules. The 5’ cap is required for increasing the stability of mRNA as it protects the mRNA from 5’ to 3’ exonuclease activity. It also is required for ribosome binding during translation as it helps recruit transcription initiation factors. It also allows 5′ to 3′ looping of mRNA during translation. As 5’ cap is added to mRNA even before transcription termination, as it is required for termination of transcription.

If there is no 5’ Cap, then the mRNA will be degraded. Such mRNA if formed also cannot translate proteins in eukaryotes.

c. GTPases are required for secretion of insulin from pancreatic beta cells. GTPase such as Rab3a and Rab27a are involved in insulin secretion. GTPase hydrolyze GTP to GDP and Pi.

It is known that movement of preproinsulin into ER requires GTP hydrolysis. Alpha subunit of SRP receptor has a GTP binding domain. GTP hydrolysis is required to transport the preproinsulin mRNA to ER and also to increase the translocation of preproinsulin to the ER lumen. Further, GTPase are required for secretion of insulin. They are also required for ER to trans-Golgi network transfer via ARF6 and other proteins.

If GTPase are not present, then there is defective processing, glycosylation and secretion of the eukaryotic insulin. In case of bacteria, there is no secretion due to lack of ER and Golgi. Bacterial insulin therefore would not be secreted but present in cytoplasm.