Site Directed Mutagenesis and Protein Engineering:

Site Directed Mutagenesis and Protein Engineering: Mutants are essential prerequisite for any genetic study in relation to study of gene structure and function Classically, mutants are generated by treating the test organism with chemical or physical agents that modify DNA called mutagens This method of mutagenesis has been extremely successful, as witnessed by the growth of molecular biology, but suffers from a number of disadvantages First, any gene in the organism can be mutated and the frequency with which mutants occur in the gene of interest can be very low. This means selection strategies have to be developed Second, even when mutants with the desired phenotype are isolated there is no guarantee that the mutation has occurred in the gene of interest Third, prior to the development of gene cloning and sequencing techniques, there was no way of knowing where in the gene, the mutation had occurred and whether it arose by a single base change, an insertion of DNA or a deletion As techniques in molecular biology have developed, so that isolation and study of single gene is not just possible but routine, so mutagenesis has also been refined

Instead of crudely mutagenizing many cells or organisms and then analyzing many thousands or millions of off springs to isolate a desired mutant, it is now possible to specifically change any given base in a cloned DNA sequence This technique is known as site-directed mutagenesis It has become a basic tool of gene manipulation for it simplifies DNA manipulations that in the past required a great deal of efforts and hard work. For example: The creation or elimination of cleavage sites for restriction endonucleases The importance of site directed mutagenesis goes beyond structurefunction relationships for the technique enable mutant protein to be generated with very specific changes in particular amino acids protein engineering Such mutants facilitates the study of mechanisms of catalysis, substrate specificity stability etc. Similarly such proteins that are better suited than naturally occurring counterparts can be created for therapeutic and industrial applications. For example:

By altering both the Michaelis constant (Km) which reflects tightness of substrate binding to enzyme and the maximal rate of conversion of the substrate in to product under defined conditions (Vmax) for an enzyme-catalyzed reaction to improve the overall catalytic efficiency (Vmax /Km) of the reaction, where Vmax equals the total amount of enzyme present (Eo) times the catalytic rate constant (kcat) by: changing the thermal tolerance or ph stability or both of a protein, enabling the altered protein to be used under conditions that would inactivate the native version modifying the reactivity of an enzyme in non-aqueous solvents so that chemical reactions can be catalyzed under non-physiological conditions changing an enzyme so that a cofactor in no longer required for certain continuous industrial production processes in which the cofactor must be supplied on regular basis modifying the substrate-binding site of an enzyme to increase its specificity, thereby decreasing the extent of undesirable side reactions increasing the resistance of a protein to cellular proteases, which would simplify purification and increase the recoverable yield altering the allosteric regulation of an enzyme to diminish the impact of metabolite feed back inhibition and increase the product yield

Site Directed Mutagenesis Methods: It is not a simple matter to produce a new protein with specified predetermined properties However, it is quite feasible to modify the existing properties of known proteins Theoretically these changes can be carried out at either the protein level or the gene level However, chemical modifications of proteins generally are harsh, nonspecific and required repeatedly, for each protein badge, so it is preferable to manipulate the DNA sequence of a cloned gene to create altered protein with novel properties With the help of computer programs, it has become a little easier to make accurate predictions of protein function on the basis of deduced amino acid sequences The process for generating amino acid coding changes at the DNA level is called as mentioned earlier site directed mutagenesis Three different methods of site-directed mutagenesis have been devised; i. Cassette ii. Primer extension iii. Procedure based on the PCR

i. Cassette Mutagenesis; In this technique, a synthetic DNA fragment containing the desired mutant sequences is used to replace the corresponding sequence in the wild type gene This method was originally used to generate improved variants of the enzyme subtilisin used in detergent It is simple method for which the efficiency of mutagenesis is close to 100 % but unique restriction site flanking the region of interest is needed The disadvantages are: i. The requirement for unique restriction sites flanking the region of interest and ii. The limitation on the realistic number of different oligonucleotide replacement which can be synthesized The later problem can be minimized by the use of doped oligonucleotides or by suppression of amber codons Fig 7.8 Primrose 6 th Ed / Fig 11.9 Primrose 5 th Ed Amber mutation is one which results in premature peptide chain termination during translation This happens because of the creation of a stop codon (UAG) in the gene sequence

Fig 11.11 Primrose 5 th Ed Bacterial strains capable of suppressing amber mutations produce trna molecules which recognize the amber codon and insert an amino acid during protein synthesis A set of E.coli strains are known to contain different amber suppressor trnas; each strain inserts a different amino acid at the amber codon This has been exploited By site directed mutagenesis an amber codon is introduced in to the desired position of the gene which then is cloned in an expression vector This vector is introduced in to each of the different suppressor strains and the expressed protein isolated fromeach one 163 different amber mutants phage T4 lysozyme have been isolated Then these mutants were introduced in to 13 suppressor strains to generate over 2000 variants of the enzyme ii. Primer Extension: The Single-Primer Method; The simplest method of site directed mutagenesis is single primer method The methods involves priming in vitro DNA synthesis with a chemically synthesized oligonucleotides (7-20 nucleotides long) that carries a base mismatch with complementary sequence

ATC

The method requires that the DNA to be mutated is available in single stranded form and cloning the gene in M13 based vectors makes this easy Fig 7.9 Primrose 6 th Ed /Fig 11.1 Primrose 5 th Ed Fig 7.10 Primrose 6 th Ed / Fig 11.2 Primrose 5 th Ed Following transformation and in vivo DNA synthesis, segregation of the two strands of the heteroduplex molecule can occur, yielding a mixed population of mutant and non-mutant progeny Clones identified as mutants require further plaque purification and identification An alternative method makes use of the observation that certain restriction enzymes like AvaI, AvaII, BanII, HindII, NciI and PvuI can not cleave phosphorothiote DNA The mutant oligonucleotide is annealed to the single stranded DNA template in the usual manner, but is then extended by DNA polymerase in the presence of thionucleotides Fig 11.3 Primrose 5 th Ed Fig 11.4 Primrose 5 th Ed iii. PCR methods: PCR method of DNA amplification showed its potential for mutagenesis

iv. Simpler and faster protocols Fig 7.11 Primrose 6 th Edition / Fig 11.5 Primrose 5 th Ed Fig 7.12 Primrose 6 th Edition / Fig 11.6 Primrose 5 th Ed Making Unidirectional Deletions; Fig 11.7 Primrose 5 th Ed v. Construction of Genes for Chimeric Proteins; vi. Fig 11.8 Primrose 6 th Ed Random Mutagenesis: This approach is a powerful way of rapidly building a structure function database in a protein system and can be equally powerful in the isolation of molecules with improved properties Random mutagenesis of gene / gene segment is best carried out suing doped oligonucleotides Fig 7.8 Primrose 6 th Ed / Fig 11.9 Primrose 5 th Ed Fig 8.5 Glick 3 rd Ed Fig 5.1 Glick 3 rd Ed Fig 5.2 Glick 3 rd Ed Fig 5.3 Glick 3 rd Ed

Phosphoramidite method DNA synthesizer / Gene machine - With acetonitrile to remove H 2 O - Column flushed with argon With TCA Acetonitrile-argon * Next prescribed base - Phosphoramidite and - Tetrazol simultaneously for *

DMT-dimethoxytrityl at 5 terminus to prevent the 5 OH from reacting non-specifically before addition of 2 nd nucleotide Controlled pore glass (uniform pore size)

Fig 8.6 Glick 3 rd Ed vii. Fig 8.7 Glick 3 rd Ed Selection of Mutant Peptides by Phage and Plasmid Display; In phage display, a segment of foreign DNA is inserted in to either a phagemid or an infectious filamentous phage genome and expressed as a fusion product with a phage coat protein It is very powerful technique for selecting and engineering polypeptides with novel functions This technique was developed first for E. coli phage M13, then extended to other phages such as T4 and λ The M13 phage particle consists of a single-stranded DNA surrounded by a coat consisting of several thousand copies of the major coat protein p8 At each end of the particle are five copies each of the two minor coat proteins p9 and p7 and at the other end five copies each of p3 and p6 In early examples of phage display, a random DNA cassette was inserted in to either the P3 or the p8 gene at the junction between the signal sequence and the native peptide E. coli transfected with the recombinant DNA molecules secreted phage particles that displayed on their surface the amino acids encoded by the foreign DNA

Particular phage displaying peptide motifs with, for example, antibody binding properties were isolated by affinity chromatography Fig 17.7 Branden Fig 7.13 Primrose 6 th Ed / Fig 11.10 Primrose 5 th Ed Fig 17. 8 Branden Protein Engineering: About 20 of the many thousands of enzymes that have been studied and characterized biochemically accounted for over 90% of the enzymes that currently being used industrially Some of the most important enzymes and their primary uses are listed in following table Table 8.12 Glick 3 rd Ed A major reason why additional enzymes are not used to any great extent in industrial processes is that an activity that have evolved to perform a particular function for a microorganism, animal or plant under natural conditions usually is not well suited for a highly specialized industrial application Most enzymes are easily denatured by exposure to the conditions, such as high temperature and the presence of organic solvents, that are used in many industrial processes

Although thermotolerent enzymes can be isolated from thermophilic microorganisms, these organisms often lack the particular enzyme that is required for use in industrial processes However, with the availability of site directed mutagenesis and gene cloning, these constraints are no longer significant Various manipulations which can be done are as follows: i. Adding Disulfide Bonds ii. Changing Asparagine to Other Amino Acids iii. Reducing the Number of Free Sulfhydryl Residues iv. Modifying Metal Cofactor Requirements v. Decreasing Protease Sensitivity vi. Modifying Protein Specificity vii. Increasing Enzyme Stability and Specificity viii. Altering Multiple Properties i. Adding Disulfide Bonds: A. T4 Lysozyme; The thermo stability of a protein can be increased by creating a molecule that will not readily unfold at elevated temperatures

In addition, these thermostable enzymes are often resistant to denaturation by: - Organic solvents and - Non-physiological conditions such as extremes of ph Addition of disulfide bonds can significantly increase the stability of protein Fig 8.13 Glick 3 rd Ed Table 8.2 Glick 3 rd Ed Fig 17.3 Branden 2 nd Ed B. Xylanase; In a similar study, the development of a temperature-stable mutant of enzyme xylanase from Bacillus circulans was undertaken During the making of paper, wood pulp is chemically bleached to remove the hemi-cellulose Unfortunately, this step results in creation of large amounts of potentially toxic effluent From an environmental perspective, treatment of wood pulp with xylanase, which degrades hemi-cellulose, is preferred to bleaching

Thermo stability is defined at temperature where 50% denaturation circular dichroism of protein in solution

A C With zero activity B pwt

Treatment of wood pulp with this enzyme could lower the amount of bleaching chemical that would otherwise be required as a part of this process However, the step at which xylanase would be added follows the hot alkali treatment of the pulp While it is possible to lower ph of this material by adding acid Current industry practice is directed toward using less water to cool the pulp, so if xylanase were to be used in this process it must function efficiently at relatively high temperatures Computer modeling of 3-dimentional structure of xylanase was used to predict sites along the polypeptide chain where one, two or three disulfide bridges could be introduced in order to stabilize without disturbing its catalytic activity All of the eight derivatives of B. circulans xylanase that were generated showed an increase in thermo stability compared with the wild type and Three of eight mutants were as enzymatically active as the wild type at 60 0 C

One mutant, in which a disulfide bridge between the N- and C-terminal ends of the enzyme was introduced, was nearly twice as active as the wild type at 60 0 C and retained more than 85% of its activity after 2 hour incubation at 60 0 C These results indicate that the thermo stability of other enzymes can be enhanced, provided that sufficiently detailed X-ray crystallographic information is available C. Human Pancreatic Ribonuclease (RNase); Ribonuclease from bull semen can act as an antitumorigenic agent In both in vitro and in vivo experiments, a dimeric form of this protein is internalized into tumor cells by nonreceptor mediated endocytosis and when it reaches the cytosol it selectively degrades ribosomal RNA, thereby blocking protein synthesis and causing cell death The dimeric form of this enzyme consists of two identical subunits covalently joined by two adjacent intersubunit disulfide bridges and stabilized by a small number of noncovalent interactions In addition, the antitumor activity of bull semen RNase is dependent on the dimeric structure of the protein, the only dimeric RNase from the pancreatic like RNase super family of proteins

However, human antibodies against bull semen RNase could be produces following repeated or prolonged use of this therapeutic protein, thereby eventually negating the usefulness and effectiveness of bull semen RNase in treating tumor cells For this reason, monomeric human pancreatic RNase was engineered to be an antitumorigenic agent The amino acid sequence of bull semen RNase is more than 70% identical to the amino acid sequence of human pancreatic RNase Accordingly, the human enzyme was engineered to become a dimer Fig 8.14 Glick 3 rd Ed When dimeric human pancreatic RNase was formed in E. coli, the protein was localized in an insoluble inclusion body Solubilization of the inclusion body and renaturation of the un folded protein yielded dimeric human pancreatic RNase that displayed a slightly lower level of antitumorigenic activity than bull semen RNase Fig 8.15 Glick 3 rd Ed

Since the dimeric human pancreatic RNase did not impair the functioning of normal diploid fibroblast cells, this engineered protein is a good candidate to become an important human therapeutic agent D. Changing Asparagine to Other Amino Acids; When proteins are exposed to high temperatures asparagine and glutamine residues may under go deamidation, an action that releases NH 3 With the loss the amide moiety, these amino acids become aspartic acid and glutamic acid respectively Resulting in localized changes in the folding of the peptide chain that may lead to a loss of activity In one study, the effect of changing some asparagine residues in Saccharomyces cerevisiae enzyme triphosphate isomerase was examined and result are given in table Table 8.3 Glick 3 rd Ed E. Reducing the Number of Free Sulfhydryl Residues; Occasionally, an expressed foreign protein is less active then expected Protein engineering can be used to increase this activity

This enzyme has two identical subunits. Each contains Asn at 14, 78 positions and may contribute towards thermo stability because they are located at the subunit interface When both asparagine residues were changed to aspartic acid residues, the resulting enzyme was unstable even at ambient temperature and had low enzymatic activity

For example, when the cdna of human β interferon (IFN-β) was cloned and expressed in E. coli, the protein product showed with only a disappointing 10% of antiviral activity of the authentic glycosylaled form Although reasonable amounts were synthesized, most of the IFN-β was found to be existed as dimers and higher oligomers that were inactive Analysis of DNA sequence revealed that native protein has three cysteine residues so one or more involved in inter molecular disulfide bonding that produced dimers and oligomers in E. coli but not in human cells It was reasoned that conversion of one or more of the cysteine codons into serine codons might result in an IFN-β derivative that would not form oligomers Serine was chosen to replace cysteine because the structures of the two amino acids are identical, except that serine contains oxygen instead of sulfur and as a result cannot form disulfide bonds When this study was undertaken, detailed information about the structure of IFN-β was lacking, so researchers were forced to rely on data derived from related proteins Fig 8.16 Glick 3 rd Ed

This deduction proved to be correct No multimeric complexes were formed when Ser-17 variant of IFN-β was expressed in E. coli Moreover, the Ser-17 IFN-β had a specific activity similar to that of authentic, native IFN-β and was more stable during long term storage than the native form F. Increasing Enzymatic Activity; In addition to stabilizing an enzyme by site-directed mutagenesis, it may be feasible to modify its catalytic function Currently, detailed information about the geometry of the active site of a well characterized enzyme is required in order to alter enzymatic activity in a meaning full way With such data researchers were able to deduce which specific changes might be necessary to modulate the substrate-binding specificity of an enzyme. For example Tyrosly- trna synthetase from B. stearothermophilus has been modified with respect to substrate binding These results are illustrated of what can be done in this area of research?

Tyrosly-tRNA synthetase catalyzes the aminoacylation of a trna that specifically accepts tyrosine (trna Tyr) in two step process: Try + ATP Tyr-A + PPi Try-A + trna tyr Tyr-tRNA tyr + AMP The 3- dimensional structure of enzyme had already been determined and active site had mapped With the aid of computer graphics, predictions were made about the effects of changing one or more amino acid residues of the active site on the interaction of the enzyme with the reaction substrates To test whether these predictions are correct, the gene of the enzyme was specifically modified by oligonucleotide directed mutagenesis The resultant enzyme variants were characterized be determining their kinetic constants and some of the observed changes were more dramatic than anticipated Table 8.4 Glick 3 rd Ed G. Modifying Metal Cofactor Requirements; Subtilisins are a group of 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) = 10 7 ] to one or more molecules of Ca 2+ per molecule of enzyme Calcium binding stabilizes the enzyme Unfortunately, since subtilisins are used in industrial settings where there are large number of metal chelating agents that can bind Ca 2+, these enzymes are rapidly inactivated under these conditions To circumvent this problem, it is initially necessary to abolish completely the ability of a subtilisin to bind Ca 2+ and then to attempt to increase stability of the modified enzyme The starting point for development of a modified subtilisin was the isolated subtilisin BNP gene from Bacillus amyloliquefaciens Prior to this work, the subtilisin BPN protein has 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 potion of protein amino acid residues 75 to 83-that is responsible for binding to Ca 2+ The protein without this stretch of amino acids does not bind Ca 2+ and surprisingly, retain an overall conformation that is similar to the native form The next steps in the development of a stable subtilisin from one that lacked a Ca 2+ binding domain entailed determining which might contribute to the stability and which amino acids should be placed in these sites The researchers assumed that any of the amino acids that had previously interacted with the Ca 2+ -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 and initial screening, stabilizing mutations were identified at 7 of the 10 positions that were examined Fig 8.17 Glick 3 rd Ed Table 8.5 Glick 3 rd Ed

H. Decreasing Protease Sensitivity; Streptokinase, a 47 kilodalton (kda) protein produced by pathogenic strains of Steptococcus 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 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 muscle Plasmin is a trypsin like protease that specifically cleaves the peptide bond after lysine or arginine residue

Plasmin rapidly digests the 41-amino acids streptokinase protein by cleaving it at lysine 59, near the N-terminus, and at lysine 386, near C-terminus 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 lysine residues at positions 59 and 386 were changed to glutamine by site directed mutagenesis Fig 8.18 Glick 3 rd Ed This work is an important first step in the development of variants of streptokinase with significantly longer half-life I. Increasing Enzyme Stability and Specificity: The enzyme tissue plasminogen activator (tpa) is a multidomain serine protease that is medically useful for dissolution of blood clots However, like strptokinase, 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

21 fold more protease resistant

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 in to the gene for the native form of tpa Table 8.6 Glick 3 rd Ed x ------------------------ x ------------------------ x --------------------------- x

- First, changing Thr-103 to Asn causes the enzyme to persist in rabbit plasma approximately 10 times longer than the native form - Second, changing amino acids 296 to 299 (variant 2) produces an enzyme that is much more specific for fibrin than is the native form - Third, changing Asn-117 to Gln causes enzyme to retain the level of fibrinolytic activity found in the native form (not shown in the table) - By combining these three mutations in a single construct allows all three activities to be expressed simultaneously - Additional work is required to determine whether a modified form of tpa is an acceptable replacement for native tpa