Molecular Biotechnology. Bernard R. Glick

and was expressed (and secreted). The selected transformants were sensitive to ampicillin, indicating that a double crossover had occurred, and that following the exchange of DNA, the plasmid was lost from the population. Resistance to ampicillin would indicate that a single recombination event had occurred and caused the integration of the entire plasmid into the B. subtilis chromosomal DNA. Integration at the correct chromosomal site may be confirmed by PCR using primers that anneal to sequences flanking the integration site.

      For cloned genes whose protein products do not have an activity that can be easily detected in a screen for transformants, a two-step procedure may be employed for chromosomal integration (Fig. 3.19). First, a selectable marker gene, usually an antibiotic resistance gene, is inserted into the middle of a nonessential piece of host cell chromosomal DNA that had previously been cloned in a plasmid. Following transformation of a bacterial host with this construct, cells expressing the antibiotic resistance gene are selected by plating on solid medium containing the antibiotic. Because the plasmid cannot replicate in the host cell, antibiotic resistant transformants have integrated the marker gene in the chromosomal DNA by homologous recombination. Next, a target gene with its transcriptional and translational control sequences is inserted into the middle of a cloned fragment of chromosomal DNA with the same sequence that flanks the marker gene and the construct is introduced into the cell, again on a nonreplicatable plasmid. Following transformation with the target gene–plasmid construct, transformants are screened for loss of the antibiotic resistance marker gene. Cells are first plated on medium that lacks the antibiotic and then colonies are replica plated onto medium containing the antibiotic to identify those that have reverted to antibiotic sensitivity. These transformants have exchanged the marker gene for the target gene, which is integrated into the chromosomal DNA.

      Figure 3.19 Insertion of a foreign gene into a unique predetermined site on the B. subtilis chromosome. In step 1, a marker gene is integrated into the host cell chromosomal DNA by homologous recombination. In step 2, the selectable marker gene is replaced by the target gene.

      When nonessential host genes have not been identified, a cloned gene may be introduced into the host chromosome by means of a single crossover that incorporates the entire input plasmid into the host chromosome (Fig. 3.20). This occurs when the cloned gene is inserted (on a plasmid) adjacent to the cloned chromosomal integration site. In this case, any selectable markers on the plasmid (including antibiotic resistance genes) will also be inserted into the host chromosomal DNA. While the integration of a selectable marker gene along with a gene of interest is helpful for identifying transformed cells, the presence of a selectable marker gene for antibiotic resistance is often undesirable, for example, for organisms that are intended for deliberate release into the environment (e.g., to degrade pollutants). To avoid the problems associated with these approaches, methods for selective removal of marker genes from host cell chromosomes have been developed. An overview of one of these methods is described here.

      Figure 3.20 Integration of a plasmid containing a cloned gene into a chromosomal site. The cloned gene is inserted adjacent to the cloned DNA from the host chromosome (c). Homologous DNA pairing occurs between plasmid DNA region c and host chromosome DNA region c′. A single recombination event (×) within the paired c–c′ DNA region results in the integration of the entire plasmid, including the cloned gene.

      When a marker gene is flanked by certain short specific DNA sequences and then inserted into either a plasmid or chromosomal DNA, the gene may be excised by a site-specific recombinase that recognizes the flanking DNA sequences and catalyzes recombination that results in excision of the intervening marker sequence (Fig. 3.21). One combination of an enzyme and DNA sequence that is useful for this sort of manipulation is the Cre–loxP recombination system, which consists of the Cre recombinase enzyme and two 34-bp loxP recombination sites. The marker gene to be removed is flanked by loxP sites, and after integration of the plasmid into the chromosomal DNA, the marker gene is removed by the Cre enzyme. A gene encoding the Cre enzyme is located on its own plasmid, which can be introduced into the chromosomally transformed host cells. Marker gene excision is triggered by the addition of IPTG to the growth medium, which turns on the E. coli lac promoter–operator that is present upstream of the cre gene and causes the Cre enzyme to be synthesized. Once there is no longer any need for the Cre enzyme, the plasmid that contains the gene for this enzyme may be removed from the host cells by raising the temperature. This plasmid has a temperature-sensitive replicon that allows it to be maintained in the cell at 30°C but not above 37°C. Given the alarming increase in antibiotic resistant strains of many bacteria, it is essential to avoid the introduction and spread of antibiotic resistant genes in the environment. Removing antibiotic resistant genes from genetically engineered bacteria is an important step in that direction.

      Figure 3.21 Removal of a selectable marker gene following integration of plasmid DNA into a bacterial chromosome. A single crossover event (×) occurs between chromosomal DNA and a homologous DNA fragment (hatched) on a plasmid, resulting in the integration of the entire plasmid into the chromosomal DNA. The selectable marker gene, which is flanked by loxP sites, is excised by the action of the Cre enzyme, leaving one loxP site on the integrated plasmid. The Cre enzyme is on a separate plasmid within the same cell under the transcriptional control of the E. coli lac promoter so that excision is induced when IPTG is added to the growth medium.

Heterologous Protein Production in Eukaryotic Cells

      Proteins from a wide variety of organisms have been successfully produced using prokaryotic expression systems. Although expression of any gene from any source organism in a prokaryotic host is theoretically possible, in practice, the eukaryotic proteins produced in bacteria do not always have the desired biological activity or stability. In addition, despite careful purification procedures, bacterial compounds that are toxic or that cause a rise in body temperature in humans and animals (pyrogens) may contaminate the final product. To avoid these problems, investigators have developed eukaryotic expression systems in fungal, insect, and mammalian cells for the production of therapeutic agents for either humans or animals; large quantities of stable, biologically active proteins for biochemical, biophysical, and structural studies; and proteins for industrial processes. Moreover, any human protein intended for medical use must be identical to the natural protein in all its properties. The inability of prokaryotic organisms to produce authentic versions of eukaryotic proteins is, for the most part, due to improper posttranslational protein processing, including improper protein cleavage and folding, and to the absence of appropriate mechanisms that add chemical groups to specific amino acid acceptor sites.

Posttranslational Modification of Eukaryotic Proteins

      In prokaryotes, the steps in protein synthesis are not compartmentalized, and therefore, translation of mRNA occurs concurrently with transcription; as soon as the nascent transcript emerges from RNA polymerase, it is accessible to the ribosome to begin translation. With the aid of folding proteins, known as chaperones, that bind to polypeptides as they are being synthesized, proteins are folded into their proper three-dimensional configuration during synthesis. In contrast, eukaryotes transport mRNA from the nucleus to ribosomes in the cytoplasm or on the endoplasmic reticulum, where translation occurs. Proteins produced on ribosomes associated with the endoplasmic reticulum either are inserted in the membrane of the endoplasmic reticulum or are secreted into the lumen of the endoplasmic reticulum during synthesis, where they are processed further.

      Many proteins, including most of those that are of interest as therapeutic agents for the treatment of human or animal diseases, undergo some type of posttranslational processing that is often required for protein activity and stability. Some proteins are produced