Molecular Biotechnology. Bernard R. Glick

to that of the native form.

      Figure 3.59 Genetic engineering of calcium-independent subtilisin. The native calcium-containing enzyme is highly active but loses almost all of its activity when the loop that binds the calcium is deleted. After several rounds of random mutagenesis, mutants of the deleted enzyme, each with stabilizing mutations and a low level of activity, are selected. Several of these mutations are combined into a single derivative with the result that a subtilisin that does not require calcium and that has a high level of activity is produced.

      The next steps in the development of a stable subtilisin from one that lacked a calcium-binding domain entailed predicting which sites might contribute to stability and which amino acids should be placed at these sites. The researchers assumed that any of the amino acids that had previously interacted with the calcium-binding loop in the native form of the enzyme were potential candidates for change. In total, 10 amino acids were considered to be candidates for modification. Moreover, since it was not known a priori which particular amino acids might best contribute to stabilizing the enzyme molecule, random mutagenesis was used for each of these sites.

      The amino acids selected for mutagenesis came from four separate regions of the protein: the N terminus (amino acids 2 to 5), the omega loop (amino acids 36 to 44), an α-helical region (amino acids 63 to 85), and a β-pleated region (amino acids 202 to 220). To identify the best amino acid at a particular position, Bacillus subtilis clones expressing the mutated proteins were grown in the wells of microtiter plates, heated to 65°C for 1 hour, allowed to cool, and then assayed for subtilisin activity. It was necessary to express the active calcium-free subtilisin in the host B. subtilis because it was lethal when expressed in E. coli.

      After the initial screening, stabilizing mutations were identified at 7 of the 10 positions that were examined (Table 3.18). When these stabilizing mutations were combined into a single gene, the enzyme that was produced had kinetic properties that were very similar to those of the native form of subtilisin. Moreover, the modified form of subtilisin was nearly 10 times more stable than the native form of the enzyme in the absence of calcium and, surprisingly, about 50% more stable than the native enzyme in the presence of calcium. This work demonstrates that complex properties of enzymes that involve a large number of different amino acids can be genetically engineered.

Table 3.18 Effects of random mutations of selected amino acid residues on the stability of a subtilisin lacking a calcium-binding domain

Decreasing Protease Sensitivity

      Streptokinase, a 47-kilodalton (kDa) protein produced by pathogenic strains of Streptococcus bacteria, is a blood clot–dissolving agent. Streptokinase forms a complex with plasminogen that results in the conversion of plasminogen to plasmin, the active protease that degrades fibrin in the blood clot. Unfortunately, plasmin also rapidly degrades streptokinase, making it necessary for medical personnel to administer streptokinase as a 30- to 90-minute infusion so that a sufficient level of intact and active streptokinase is maintained. Since it is essential that individuals suffering a heart attack be treated as quickly as possible, a long-lived streptokinase could be administered as a single injection before a person is transported to a hospital. This early treatment might contribute to saving the lives of heart attack victims by quickly restoring blood flow and limiting damage to heart muscles.

      Plasmin is a trypsin-like protease that specifically cleaves the peptide bond after a lysine or arginine. Plasmin rapidly digests the 414-amino-acid streptokinase protein by cleaving it at lysine 59, near the N terminus, and at lysine 386, near the C terminus (Figure 3.60A). The 328-amino-acid peptide that remains following the digestion by plasmin has approximately 16% of the activity of intact streptokinase in activating plasminogen, and it is slowly degraded by plasmin until no activity remains. To make streptokinase less susceptible to proteolysis by plasmin, the lysines at positions 59 and 386 were changed to glutamine by site-directed mutagenesis (Fig. 3.60B-D). Glutamine was chosen to replace lysine because the length of its side chain is similar to that of lysine, so that the three-dimensional structure would not be disturbed, and because glutamine does not have a positive charge. Both single mutants, as well as the double mutant, had the same ability to bind to and activate plasminogen as did the native form of streptokinase. At the same time, in the presence of plasmin, the half-lives of all three mutants were increased compared with native streptokinase, with the double mutant being approximately 21-fold more protease resistant. This work is an important step in the development of variants of streptokinase with significantly longer half-lives.

      Figure 3.60 Protease (plasmin) sensitivity of streptokinase and some engineered plasmin-resistant derivatives. The green circles indicate positively charged lysines where plasmin cuts the polypeptide. The red circles indicate glutamines where plasmin does not cut the polypeptide. The horizontal arrows indicate plasmin digestion of streptokinase. The protein size and activity following plasmin digestion are indicated for each derivative. (A) Native protein; (B) the derivative in which glutamine replaces lysine 386; (C) the derivative in which glutamine replaces lysine 59; (D) the derivative in which glutamines replace lysine 59 and lysine 386.

summary

      The production of a protein requires that the gene be properly transcribed and then that the mRNA be translated. In prokaryotes, a promoter sequence is necessary for the initiation of transcription at the correct nucleotide site, and a terminator sequence at the end of the gene is essential for the cessation of transcription. The aim of many biotechnology applications is to produce large amounts of protein, so it is necessary to use a strong promoter that supports transcription at a high level such as the promoter from gene 10 of the E. coli bacteriophage T7. However, continuous transcription of a cloned gene drains the energy reserves of the host cell; therefore, it is also necessary to use a promoter system whose activity can be regulated, such as the E. coli lac promoter that is induced by addition of lactose or IPTG. For translation, a ribosome-binding site is placed in the DNA region that precedes the translation initiation site (start codon), and a termination sequence (stop codon) is included at the end of the protein coding sequence to ensure that translation stops at the correct amino acid. Codon optimization may be required for production of foreign proteins in some host cells. If secretion of the protein is desired, the DNA sequence preceding the cloned gene should include a signal sequence in the same reading frame as the target gene. In addition, amino acid purification tags are added to purify the recombinant protein by, for example, immunoaffinity chromatography. In these cases, the junction point of a fusion protein is usually designed to be cleaved in vitro either chemically or enzymatically.

      High levels of expression of some foreign proteins in bacterial hosts can result in misfolded proteins that form insoluble inclusion bodies. This can be avoided by growing the recombinant bacterial strains at low temperatures, coexpressing chaperone proteins, overexpressing enzymes that catalyze the formation of disulfide bonds, and/or expressing the target protein as a fusion protein. Recombinant proteins may also be degraded by proteolytic enzymes synthesized by the host cell. To overcome this problem, a cloned gene is altered to encode one or more additional amino acids at its N terminus or to remove protease recognition sequences. In this form, the recombinant protein is no longer rapidly degraded. During large-scale production of recombinant proteins, plasmids may be unstable and lost from the population. To overcome this problem, researchers have developed protocols for integrating a cloned gene into a chromosomal site of the host organism. Under these conditions, the gene is maintained stably as part of the DNA of the host organism.

      Although many heterologous proteins have been successfully synthesized in prokaryotic host cells, some proteins require eukaryote-specific posttranslational modifications, such as glycosylation, to