Molecular Biotechnology. Bernard R. Glick

to prevent histidine protonation at low pH that causes RAP to unfold and dissociate from LRP1 (Fig. 3.56C). The two sets of mutations, that is, the introduction of the cysteines and the elimination of the histidines, were introduced into RAP separately and in combination. At pH 7.4, the wild-type and mutant RAP proteins were properly folded (measured as the portion of α-helical content of the protein). At pH 5.5, wild-type RAP unfolded and its affinity for LRP1 was reduced 181-fold (Table 3.16). In contrast, at low pH, mutant RAP with the disulfide bond and mutant RAP with histidines replaced with phenylalanine were substantially folded. Moreover, RAP with the combined mutations remained properly folded. Importantly, the LRP1 binding affinity of the mutant RAP proteins at pH 5.5 was reduced only 22- to 53-fold compared to that at pH 7.4 (Table 3.16). Treatment of LRP1-expressing human fibroblast cells with combined mutant RAP significantly inhibited ligand (α2-macroglobulin) uptake compared to treatment with wild-type RAP, indicating that recycling of the receptor was prevented to a greater extent by the acid-stable, mutant RAP. A similar result was found in mice. This study shows that engineering RAP for increased stability generates a more potent inhibitor of LRP1 than wild-type RAP and is a suitable treatment for diseases mediated by LRP1 clearance of blood proteins.

      Figure 3.56 Site-directed mutagenesis of receptor associated protein (RAP) to increase acid stability. (A) Following binding of exogenous RAP to low-density lipoprotein receptor-related protein 1 (LRP1) in the cell membrane, the protein complex is taken into the cell by endocytosis. Acid-sensitive wild-type RAP is denatured in the acidic endosome and releases LRP1, which is recycled back to the cell membrane. (B) To increase the acid-stability of RAP, a disulfide bond was introduced into domain D3. Tyrosine at position 260 (Y260) and threonine at position 297 (T297) were changed to cysteines (C260 and C297) by site-directed mutagenesis. (C) To further increase acid-stability, four histidines (H257, H259, H268, H290) that are protonated at low pH were changed to phenylalanine (F257, F259, F268, F290). Adapted from Prasad et al., 2015. J. Biol. Chem. 290:17262.

Table 3.16 Binding affinity of LRP1 for mutant RAP

Modifying Protein Specificity

      The enzyme tissue plasminogen activator (tPA) is a multidomain serine protease that is medically useful for the dissolution of blood clots. However, tPA is rapidly cleared from the circulation, so that it must be administered by infusion. Therefore, to be effective with this form of delivery, high initial concentrations of tPA must be used. Unfortunately, under these conditions, tPA can cause nonspecific internal bleeding. Thus, a long-lived tPA that has an increased specificity for fibrin in blood clots and is not prone to induce nonspecific bleeding would be desirable. It was found that these three properties could be separately introduced by site-directed mutagenesis into the gene for the native form of tPA. First, changing threonine at position 103 (Thr-103) to asparagine (Asn) causes the enzyme to persist in rabbit plasma approximately 10 times longer than the native form (Table 3.17). Second, changing the amino acids lysine-histidine-arginine-arginine (Lys-His-Arg-Arg) at 296 to 299 to alanines (Ala-Ala-Ala-Ala) produces an enzyme that is much more specific for fibrin than is the native form. Third, changing Asn-117 to glutamine (Gln) causes the enzyme to retain the level of fibrinolytic activity found in the native form. Moreover, combining these three mutations in a single construct allows all three activities to be expressed simultaneously (Table 3.17).

Table 3.17 Stabilities and activities of various modified versions of tPA

      Using random mutagenesis, it is possible to generate antibodies in vitro that are directed against a wide range of antigens. The portion of an antibody molecule that contains the ability to bind to an antigen is sometimes called a Fab fragment, and within this fragment are hypervariable complementarity-determining regions (CDRs) separated by relatively invariant framework regions (Fig. 3.57). Together, the six CDRs, three from the variable part of the light chain and three from the variable part of the heavy chain (see chapter 4), determine the specificity of an antibody molecule. Altering one or more of the amino acids in one of the CDRs changes the specificity of the antibody.

      Figure 3.57 Structure of a Fab molecule. FR, framework region; CDR, complementarity-determining region. CH1 and CL are constant domains from the heavy and light chains of the antibody molecule, respectively. The N-terminal (NH2) and C-terminal (COOH) ends of each polypeptide, as well as a disulfide bridge (-S-S-), are indicated.

      Using degenerate oligonucleotide primers, it was possible to introduce a range of different mutations into the three CDRs of the variable region of an antibody heavy-chain gene (Fig. 3.58). First, one of the CDRs was modified by PCR. Then, in a second PCR, the other two CDRs were modified. Finally, the three altered CDRs were combined in a single DNA fragment. The same changes can also be introduced into the gene for the variable portion of an antibody light chain. Using this approach, a Fab fragment of a monoclonal antibody that was specific for the compound 11-deoxycortisol was altered to produce a Fab fragment that was specific for cortisol and no longer bound 11-deoxycortisol. In theory, Fab fragments directed against any antigen can be generated with this method.

      Figure 3.58 Random mutagenesis used to introduce mutations into the three CDR genes of the variable region of a heavy antibody chain. The framework region sequences are shown in green, and the CDR sequences are in blue. (A) The first PCR with a degenerate forward primer (top arrow) introduces random mutations into the DNA encoding CDR1. (B) The second PCR with degenerate primers introduces random mutations into the DNA encoding CDR2 and CDR3. (C) The third PCR combines the DNA that was amplified in panels A and B. The circled portion of the DNA indicates the place where random mutations were introduced.

Modifying Cofactor Requirements

      Subtilisins are a group of nonspecific serine proteases that are secreted into growth medium by Gram positive bacteria and are widely used as biodegradable cleaning agents in laundry detergents. All subtilisins bind tightly (affinity constant [Ka] = ∼107 M) to one or more molecules of calcium per molecule of enzyme where calcium binding stabilizes the enzyme. Unfortunately, since subtilisins are used in industrial settings where there are a large number of chelating agents that can bind to and effectively remove calcium, these enzymes are rapidly inactivated under these conditions. To circumvent this problem, it is necessary first to abolish completely the ability of a subtilisin to bind calcium and then to attempt to increase the stability of this modified enzyme in the absence of bound calcium.

      The starting point for the development of a modified subtilisin was an isolated subtilisin gene from Bacillus amyloliquefaciens. Prior to this work, the subtilisin protein had been well characterized, and its high-resolution X-ray crystallographic structure had been determined. Oligonucleotide-directed mutagenesis was used to construct a mutant form of the gene for this enzyme by deleting the nucleotides encoding the portion of the protein—amino acids 75 to 83—that is responsible for binding to calcium (Fig. 3.59). The protein without this stretch of amino acids does not bind calcium and, surprisingly, retains an overall conformation that is similar