Molecular Biotechnology. Bernard R. Glick

      Some biologically important proteins, such as α-interferon (IFN-α), are encoded by a family of several related genes, with each protein having slightly different biological activity. If all, or at least several, of the genes or cDNAs for a particular protein have been isolated, it is possible to recombine portions of these genes or cDNAs to produce hybrid or chimeric forms (Fig. 3.52). This “DNA shuffling” is done with the expectation that some of the hybrid proteins will have unique properties or activities that were not encoded in any of the original sequences. Also, some of the hybrid proteins may combine important attributes of two or more of the original proteins (e.g., high activity and thermostability).

      Figure 3.52 Amino acid changes may be introduced into a protein by either random mutagenesis or error-prone PCR, both of which cause single-amino-acid substitutions, and by DNA shuffling, in which genes are formed with large regions from different sources.

      The simplest way to shuffle portions of similar genes is through the use of common restriction enzyme sites (Fig. 3.53). Digestion of two or more of the DNAs that encode the native forms of similar proteins with one or more restriction enzymes that cut the DNAs in the same place, followed by ligation of the mixture of DNA fragments, can potentially generate a large number of hybrids. For example, two DNAs, each of which has three unique restriction enzyme sites, can be recombined (shuffled) to produce 14 different hybrids in addition to the original DNA (Fig. 3.53).

      Figure 3.53 The 14 different hybrid genes that can be generated by combining restriction enzyme fragments from two genes from the same gene family that have three different restriction sites in common. RE, restriction enzyme.

      Another way to shuffle DNA involves combining several members of a gene family, fragmenting the mixed DNA with deoxyribonuclease I (DNase I), selecting smaller DNA fragments, and amplifying these fragments by PCR (Figure 3.54). During PCR, gene fragments from different members of a gene family cross-prime each other after DNA fragments bind to one another by complementary base pairing in regions of high homology. The final full-length products are amplified by PCR using terminal primers. After 20 to 30 PCR cycles, a panel of hybrid (full-length) DNAs will be established (Fig. 3.54). The hybrid DNAs are then cloned to create a library that can be screened for the desired activity. Although DNA shuffling works well with gene families—it is sometimes called molecular breeding—or with genes from different families that nevertheless have a high degree of homology, the technique is not especially useful when proteins have little or no homology. Thus, the DNAs must be very similar to one another or the PCR will not proceed. To remedy this situation and combine the genes of dissimilar proteins, several variations of the DNA-shuffling protocol have been described.

      Figure 3.54 Some of the hybrid DNAs that can be generated during PCR amplification of three members of a gene family.

      One procedure that was developed to combine the genes of dissimilar proteins and that does not rely on PCR amplification of DNA fragments is called nonhomologous random recombination. In this procedure (Fig. 3.55), DNAs from different sources (either defined or random DNA sequences, or a mixture of both) are combined and then partially digested with DNase I. These DNA fragments, which include a wide variety of sizes, are made blunt ended by digestion with the enzyme T4 DNA polymerase. This enzyme both fills in 5′ overhanging nucleotides and degrades 3′ overhanging nucleotides. The DNA fragments are then mixed with a synthetic DNA fragment that forms a hairpin loop and contains a specific restriction enzyme site. The entire mixture is ligated by the addition of the enzyme T4 DNA ligase that results in the formation of extended mosaic DNA molecules of variable lengths with a hairpin at each end. Ligation of the hairpins prevents further addition of fragments (concatemerization) to the molecules. The average length of these hairpin structures is dictated by the ratio between the blunt-ended DNA and the DNA hairpins added to the ligation reaction. Finally, restriction enzyme digestion removes the hairpin loops so that the resulting sticky-ended DNA fragments can be inserted into plasmid vectors and tested for various activities. Because this process randomly recombines DNA fragments, only a very small fraction of the recombined DNAs are likely to encode the desired activity.

      Figure 3.55 Nonhomologous random recombination. Different DNAs (shown in different colors) are mixed together, partially digested with DNase I, blunted at the ends by digestion with T4 DNA polymerase, size fractionated, ligated with synthetic hairpin DNAs to form extended hairpins, restriction enzyme digested to remove the hairpin ends and generate sticky ends, and then ligated into plasmid vectors.

Examples of Protein Engineering

      Increasing Protein Stability

      Proteins have evolved to perform a particular function for a microorganism, animal, or plant under natural conditions and are often not well suited for a highly specialized biotechnology application. For example, most enzymes are easily denatured by the high temperature and the presence of organic solvents that are used in some industrial processes. Although thermotolerant enzymes can be isolated from thermophilic microorganisms, these organisms often lack the particular enzyme that is required for an industrial processes. Directed mutagenesis can be used to create a protein that will not readily unfold under the conditions in which it will be employed. The addition of disulfide bonds, through the introduction of specifically placed cysteines, can usually significantly increase the stability of a protein (Fig. 3.8). Extra disulfide bonds may perturb the normal functioning of a protein and therefore the activity, as well as the stability, of a modified protein must be tested.

      In one example, a receptor antagonist protein was engineered for increased stability to enhance its effectiveness as a therapeutic agent. Low-density lipoprotein receptor-related protein 1 (LRP1) is a cell surface signaling protein that binds lipoproteins and other ligands and removes them from the bloodstream. The receptors also remove blood coagulation proteins, which leads to bleeding episodes in individuals with hemophilia. Following synthesis in the endoplasmic reticulum, LRP1 is escorted to the Golgi by a chaperone protein, receptor-associated protein (RAP). RAP is denatured in the acidic environment of the Golgi, thereby releasing the receptor proteins for subsequent processing and transport to the cell membrane. Exploiting its high affinity for LRP1, RAP can also be administered exogenously to inhibit binding of blood coagulation proteins to the receptors and thereby prevent bleeding episodes in hemophiliacs.

      Following administration of RAP, the LRP1-RAP complex that forms at the cell membrane is taken up by the cell in an endosome which results in low pH-induced denaturation of RAP and recycling of LRP1 back to the cell surface (Fig. 3.56A). Thus, the acid sensitivity of RAP limits its potential as a therapeutic agent. Researchers reasoned that introduction of a disulfide bond would increase the acid stability of RAP and thereby prevent its dissociation from LRP1. The structure of RAP is known and computer modeling was used to predict optimal sites for introduction of cysteines. Using site-directed mutagenesis, the coding sequences for two amino acids in domain D3 of RAP, a tyrosine at position 260 and a threonine at position 297, were altered to encode cysteines (Fig. 3.56B). In addition, four histidines in domain D3 were changed