Basic principles of recombinant DNA technology

by Gonzalo C. Serafica
quiz and text A. R. Deangelo (1988)

In recent decades, biochemical engineering became a major area for growth because of one single development - the ability to splice together DNA molecules from different sources to create a novel combination, termed as recombinant DNA technology

This technology has made it possible to synthesize foreign proteins such as human hormones in micorganisms. In this tutorial, you should learn how this is being done. Before we start, I would like to give you a short diagnostic test for either review or learning terminology. It is very important to remember what these terms mean because we meet them several times as we go along the steps in producing recombinant proteins.

(animation will go here) These are the sticky ends generated by cleaving the plasmid and the foreign protein using restriction endonucleases before annealing them

As an illustration on how the process of inserting the foreign DNA into the plasmid, 1)the DNA of the protein and the plasmid are cleaved separately by the same enzyme like ecori to give 4 sticky ends. Then, one end of the plasmid is joined to the complementing end on the DNA of the protein while the other two ends does likewise. This step is called the annealing. Thus, the recombinant DNA is formed.

Plasmids being used as cloning vectors must possess the following features:

  • the DNA of the cloning vector must have only a single target site for any particular restriction enzyme to avoid rearrangement or undesired loss of fragments of the plasmid. Also the more restriction endonuclease for which the plasmid has unique sites, the better it will be as a vector.
  • it must have one or more readily selectable genetic markers such as a antibiotic resistance.
  • it must have an origin of replication that ensures that they are propagated in the desired host cell. bifunctional vectors that have more than one origin of replication can be constructed and can be expressed in two separarte cells. There are three steps involved in the production or expression of a desired protein from a another organism once the recombinant DNA plasmid has been inserted into the host cell.these are
    1. transcription of DNA to mRNA
    2. translation of the mRNA into a polypeptide sequence.
    3. in some cases, post translational modification like glycosylation.
    However, in many eukaryotes, there is an additional step before the translation process, that is the removal of the non-coding sequences, the introns, from the mRNA by a process known as splicing. The transcription process starts with the mediating RNA polymerase binding to the promoter on the DNA of the desired protein. After binding, the RNA polymerase molecule travels along the DNA molecule until a termination signal is encountered. It follows that a gene that does not lie in between the promoter and a termination signal will not be transcribed.

    The translation of mRNA into protein is a complex process that involves interaction of the mRNA with the ribosomes. Similar to trancription, the mRNA must carry a ribosomal binding site (rbs) in front of the gene to be translated. After binding, the ribosome moves along them RNA and initiates protein sythesis at the first AUG codon it encounters and continues until it encounters a stop codon (UAA, UAG or UGA). If the cloned gene lacks the rbs, it is necessary to use a vector in which the gene can be inserted downstream from both a promoter and a rbs, in the direction of coding. Most of the proteins being expressed by rDNA technology undergo post-translational modifications and proteins destined to be transported out of the cell are usually synthesized with an extra 15-30 amino acids at amino-end (n-terminus) refferred to assignal sequence. These sequences should be incorporated at the time of gene construction and they will facilitate excretion of the protein product into the growth medium that is usually desirable for easy product isolation. Other possible modifications are

  • synthesizing the protein first in proprotein form for certain reasons like protection from proteolytic attack, and then only after separation is the protein properly activated.
  • glycosylation or the addition of oligosaccharides to certain amino acid residues.

    There are two important considerations in applying rDNA technology, first, properly selecting the organisms that contains the recombinant DNA plasmid from a population of the host cell, and second, maximizing the amount of gene produced from an expression system.

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