Molecular Biotechnology. Bernard R. Glick

etch virus protease, thrombin, and factor Xa. However, following this cleavage, it is necessary to perform additional purification steps in order to separate both the protease and the cleaved peptide from the protein of interest.

      Figure 3.15 Proteolytic cleavage of a fusion protein by blood coagulation factor Xa. (A) The factor Xa recognition sequence (Xa linker sequence) lies between the amino acid sequences of two different proteins. A functional target protein (with valine at the N terminus) is released after cleavage. (B) A tripartite fusion protein including a stable fusion partner, a Xa linker peptide, and the target protein.

      To avoid additional purification steps that are costly and may reduce yields of target protein, self-cleaving purification tags can be used. An intein (intervening protein) is an amino acid segment found in some natural proteins that can excise itself and join the flanking segments (exteins) with a peptide bond. Excision is mediated by a cysteine or serine amino acid at the N-terminal end of the intein sequence, an asparagine at the C-terminal end, and a cysteine, serine or threonine immediately after the asparagine (Fig. 3.16). For affinity tag removal, the target protein can be expressed with an intein-affinity tag sequence at either the N or C terminus. For removal of an N-terminal tag, the codon for the intein N-terminal cysteine/serine is replaced with the codon for alanine to prevent rejoining of the affinity tag and target protein sequences that flank the intein (Fig. 3.17A). Similarly, for cleavage of a C-terminal tag, the codon for the intein C-terminal asparagine is replaced with that for alanine (Fig. 3.17B). Following purification of a target protein, cleavage of the intein-affinity tag is induced by increasing the pH of the solution or by treatment with a thiol reagent such as dithiothreitol or β-mercaptoethanol (Fig. 3.17).

      Figure 3.16 Self-splicing proteins. Excision of an intein sequence from a precursor protein is facilitated by specific amino acids at the N- and C-end of the intein. Following excision, the N- and C-exteins are joined to produce the mature protein. C, cysteine; S, serine; N, asparagine; T, threonine. Adapted from Elleuche and Pöggler, 2010. Appl. Microbiol. Biotechnol. 87:479–489.

      Figure 3.17 Removal of protein purification tags using inteins. Affinity tags can be removed by inserting the coding sequence for an intein between the coding sequences for the affinity tag and the target protein. Following translation and purification, self-excision of the intein is induced by increasing pH or addition of a thiol reagent such as dithiothreitol. (A) Cleavage of an N-terminal tag. (B) Cleavage of a C-terminal tag. A, alanine; N, asparagine; C, cysteine. Adapted from Fong et al., 2010. Trends Biotechnol. 28:272–279.

DNA Integration into the Host Chromosome

      A plasmid vector imposes a metabolic load on the cell because of the energy that is used for its replication and for the transcription of RNA and translation of the proteins that it encodes. As a consequence, a fraction of the cell population often loses its plasmids during cell growth. Cells that lack plasmids generally grow faster than those that retain them, so plasmidless cells eventually dominate the culture. After a number of generations of cell growth, the loss of plasmid-containing cells diminishes the yield of the cloned gene product. On a laboratory scale, plasmid-containing cells are maintained by growing the cells in the presence of either an antibiotic or an essential metabolite that enables only plasmid-bearing cells to thrive. But the addition of either antibiotics or metabolites to pilot plant- or industrial-scale fermentations can be extremely costly, and it is imperative that anything that is added to the fermentation be completely removed before the product is approved for human use. For genetically engineered microorganisms that are designed to be released into the environment to remain both effective and environmentally safe, it is essential that the cloned DNA be retained and be neither easily lost nor transferred to other microorganisms. The introduction of cloned DNA directly into the chromosomal DNA of the host organism can overcome the problem of plasmid loss or transfer. When DNA is part of the host chromosomal DNA, it is relatively stable and consequently can be maintained for many generations in the absence of selective agents.

      The chromosomal integration site of a cloned gene must not be within an essential coding gene. Consequently, the input DNA sequence must be targeted to a specific nonessential site within the chromosome. In addition, to ensure efficient production of the target protein, the input gene should be under the control of a regulatable promoter.

      Chromosomal integration of the input DNA occurs by homologous recombination. For this, the input DNA must be flanked by sequences that share similarity, at least 50 nucleotides, with the sequence at the integration site. Recombination, mediated by specific host enzymes, results in physical exchange between the two DNA molecules. Briefly, a generalized protocol for DNA integration includes the following steps.

      1 Identify the desired chromosomal integration site (i.e., a segment of DNA on the host chromosome that can be disrupted without affecting the normal functions of the cell).

      2 Clone the sequence from the chromosomal integration site into a plasmid vector. The chromosomal sequence may be obtained by PCR (described in chapter 2). The plasmid vector must not have an origin of replication that enables it to be replicated in the host bacterium.

      3 Ligate a target gene and a regulatable promoter into the cloned chromosomal integration site on the plasmid (Fig. 3.18).

      4 Transfer the plasmid containing the chromosomal integration fragment–cloned-gene construct into the host cell, usually by conjugation (described in chapter 2).

      5 Select and perpetuate host cells that express the cloned gene. Propagation of the cloned gene can occur only if it has been integrated into the chromosomal DNA of the host cell.

      Figure 3.18 Integration of a cloned gene into a chromosomal site. The cloned gene has been inserted, on a plasmid, in the middle of a cloned segment of DNA (ab) from the host chromosome. Homologous DNA pairing occurs between plasmid-borne DNA regions a and b and host chromosome DNA regions a′ and b′, respectively. A double-crossover event (×) results in the integration of the cloned gene.

      When a host cell is transformed with a nonreplicating plasmid that carries the cloned gene in the middle of a portion of the cloned chromosomal integration site, the DNA on the plasmid can base-pair with complementary sequences in the homologous region of the host chromosome (Fig. 3.18). The integration occurs as a result of a host enzyme-catalyzed double crossover. Alternatively, a single crossover that incorporates the entire input plasmid into the host chromosome may occur (not shown).

      This protocol is exemplified by the integration of the α-amylase gene from Bacillus amyloliquefaciens into the chromosome of B. subtilis. The α-amylase gene, under the control of a B. subtilis promoter, was inserted into the middle of a chromosomal DNA fragment from B. subtilis that had been cloned in an E. coli plasmid. The plasmid carries the bla gene that confers resistance to ampicillin and cannot replicate in B. subtilis. The plasmid can, however, transform B. subtilis. Transformants expressing α-amylase, an enzyme involved in the hydrolysis of starch, were identified by a zone of clearance around colonies that grew on solid medium containing starch. This indicated that the α-amylase gene had been integrated into the B.