Expression vectors

Halki Diabetes Remedy

Alternative Ways to Treat Diabetes

Get Instant Access

Sometimes, the aim of a cloning experiment is not just to obtain large amounts of a specific gene, but for the gene to be expressed. This involves using the host cell as a sort of 'factory', to manufacture the specific protein encoded by the cloned gene. One of the earliest applications of genetic engineering was the production of human insulin in E. coli (Figure 12.10). Insulin is needed in considerable quantities for the treatment of diabetics; for years it was obtained from the pancreas of pigs and cattle, but this had several disadvantages including immunological complications and the risk of viral contamination. Insulin generated by recombinant means is free from these problems. Many proteins can now be produced in this way by microorganisms at a rate several times that of the normal host cell. In order for a gene to be expressed, it must have specific nucleotide sequences around it that act as signals for the host cell's transcription/translation machinery (promoter, ribosomal binding site and terminator - see Chapter 11). Since these sequences differ between, say, humans and E. coli, the bacterial RNA polymerase will not recognise the human sequences, and therefore, although a human gene may be cloned in E. coli using a simple vector, it will not be expressed. If, however, the human gene could be inserted so that it was under the control of the E. coli expression signals, then transcription should take place. Specially designed vectors that provide these signals are called expression vectors. The choice of promoter sequence is particularly important; often, a strong (i.e. very efficient) promoter is selected, so as to maximise the amount of protein product obtained. Genes whose protein products are naturally synthesised in abundance are likely to have such promoters. It is often helpful to be able to regulate gene expression; inducible promoters can be switched on and off by the presence of certain substances. The lac promoter (which controls the lacZ gene) is an example of this. Cassette vectors have promoter, ribosomal binding site and terminator sequences clustered together as a discrete unit, with a single recognition site for one or more REs being situated downstream of the promoter (Figure 12.11).

The small size of the insulin molecule (and gene) and the size of the potential market made it a prime early candidate for production by recombinant DNA technology. Most insulin used in the treatment of diabetes nowadays is produced in this way. Systems based on E. coli have also been used to synthesise other small human proteins with therapeutic potential such as human growth hormone, Y-interferon and tumour necrosis factor (TNF).

Bacteria, however, are not suitable host cells for the production of many other human proteins such as tissue plasminogen activator (TPA) or blood clotting Factor VIII, due to the size and complexity of their genes. This is because many proteins of complex eucaryotes are subject to post-translational modifications; this does not occur in procaryotes, so bacteria such as E. coli are not equipped with the cellular machinery to make the necessary modifications to human proteins.

The polypeptide products of translation in eucaryotes may require post-translational modification before functional protein is produced. Examples include phos-phorylation, acetylation and glycosylation.

Figure 12.10 Production of recombinant human insulin. Human insulin comprises two short peptide chains, 21 and 30 amino acid residues in length. Because they are so short, the nucleotide sequence of their genes can be predicted from their amino acid sequence, and synthetic genes produced. Each is cloned under the influence of the lac promoter and downstream of part of the lacZ gene. By being produced as fusion proteins with ^-galactosidase, the insulin chains are protected against being degraded by the E. coli cell. From Brown, TA: Gene Cloning, 3rd edn, Chapman & Hall, 1995. Reproduced by permission of Blackwell Publishing Ltd.

Figure 12.10 Production of recombinant human insulin. Human insulin comprises two short peptide chains, 21 and 30 amino acid residues in length. Because they are so short, the nucleotide sequence of their genes can be predicted from their amino acid sequence, and synthetic genes produced. Each is cloned under the influence of the lac promoter and downstream of part of the lacZ gene. By being produced as fusion proteins with ^-galactosidase, the insulin chains are protected against being degraded by the E. coli cell. From Brown, TA: Gene Cloning, 3rd edn, Chapman & Hall, 1995. Reproduced by permission of Blackwell Publishing Ltd.

Ribosomal Multiple binding site cloning site

Ribosomal Multiple binding site cloning site

Figure 12.11 A cassette vector. In order for a foreign gene to be transcribed and translated successfully, it must be surrounded by the appropriate expression sequences. In a cassette vector, these sequences flank the RE recognition sites at which the foreign DNA is inserted

Another obstacle to the cloning of such proteins concerns a fundamental difference in the way that procaryotic and eucaryotic systems convert the message encoded in DNA to messenger RNA (see Chapter 11). Procaryotes lack the means to remove introns, so if a human gene, for example, is expressed, the whole of the primary transcript will be translated, instead of just the coding sequences, leading to a non-functional protein. This problem can be circumvented by cloning not the entire gene, but its cDNA, that is, just those DNA sequences that are transcribed into mRNA and subsequently translated into amino acid sequences. This can be done by isolating mRNA, then using reverse transcriptase to make a DNA copy. In the case of proteins such as insulin, the very small size enabled artificial genes to be synthesised, based on their known amino acid sequences.

Complementary DNA (cDNA) is DNA produced using mRNA as a template, using the enzyme reverse transcrip-tase. It represents only the coding sequences of the parent DNA molecule.

Was this article helpful?

0 0
Diabetes Sustenance

Diabetes Sustenance

Get All The Support And Guidance You Need To Be A Success At Dealing With Diabetes The Healthy Way. This Book Is One Of The Most Valuable Resources In The World When It Comes To Learning How Nutritional Supplements Can Control Sugar Levels.

Get My Free Ebook


Post a comment