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1 Site Directed Mutagenesis and Protein Engineering BC35C Biotechnology I (Lecture notes 2004) Prepared and presented by Dr. Marcia E. Roye Office: Biotechnology.

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Presentation on theme: "1 Site Directed Mutagenesis and Protein Engineering BC35C Biotechnology I (Lecture notes 2004) Prepared and presented by Dr. Marcia E. Roye Office: Biotechnology."— Presentation transcript:

1 1 Site Directed Mutagenesis and Protein Engineering BC35C Biotechnology I (Lecture notes 2004) Prepared and presented by Dr. Marcia E. Roye Office: Biotechnology Centre, Ground floor Tel: 927-0304/977-1828 (ext. 2518-20) Email:

2 2 Lecture Objectives The objectives of these lectures are: 1.Investigate how desired mutations can be introduced into a cloned gene. 2.Explain how these mutations can be used to introduce desired properties in a protein.

3 3 Course Outline Site-directed mutagenesis and protein engineering Definitions of mutation, directed mutagenesis and protein engineering. Directed mutagenesis methods using M13, plasmid, PCR, and random. Protein engineering, introduction. What characteristics of protein are desirable? Improving protein stability by adding S-H bonds (lysozyme, xylanase, human pancreatic RNase), changing labile amino acids (triose phosphate isomerase), reducing the # of free S-H groups ( interferon). Increasing enzyme activity (tyrosyl tRNA synthase). Modifying cofactor requirement (subitilisins), increasing specificity (t plasmogen activator), decreasing protease sensitivity (streptokinase). Recommended reading: *Molecular Biotechnology, Glick, B.R. and Pasternak, J.J. *Journal References: Proceedings National Academy of Sciences (1994), 91:3670: (1984) 81:5662, (1978), 84:675.Trends in Biotechnology (1990), 8:16 Biotechnology (1995), 13:669, Protein Engineering, (1986), 1:7, 1994, 7:1379, Nature (1989), 342:291, Biotechniques, (1987), 5:786, Science (1983) 219:666. *This text and these journal articles are available in Dr Royes book rental scheme.

4 4 Getting notes from Web

5 5 Definitions Mutation: a change in the nucleic sequence (bases) of an organisms genetic material (a change in the genetic material of an organism). Directed mutagenesis: a change in the nucleic acid sequence (or genetic material) of an organism at a specific predetermined location.

6 6 Protein Engineering Protein engineering involves the use of genetic manipulations to alter the coding sequence of a (cloned) gene and thus modify the properties of the protein encoded by that gene. This mutant gene maybe expressed in a suitable system to produce unlimited quantities of the modified protein. Therefore site directed mutagenesis and protein engineering are used to change ( modify) the properties of a protein.

7 7 What Properties of a Protein Would You Want to Change? We may be able to alter: Michaelis constant Km Vmax Thermal stability pH stability Cofactor requirement Specificity Sensitivity

8 8 Km/Vmax What is the Km of an enzyme ? Michaelis constant or Km is the tightness of the substrate binding to the enzyme. (increases the specificity of the reaction and reduce side reactions). The Vmax is the maximal rate of conversion of the substrate to the products. ( an increase in Vmax increase the amount of product produced). An increase in pH or thermal stability may allow the protein to be used under conditions where it would normally be denatured.

9 9 Cofactor Requirement and Increase Specificity The abolishment of the need for a cofactor may be beneficial where under certain industrial conditions a cofactor has to be constantly provided. Increase specificity of the enzyme decreases undesirable side reactions.

10 10 The Possibilities Recombinant DNA technology has made it possible to isolate and modify any desired gene. What is recombinant DNA technology? It is not always possible to produce a completely new protein with the desired properties. But it maybe possible to through: Directed mutagenesis and Protein engineering To modify an existing protein to produce an altered protein with the desired properties.

11 11 Why Modify the Gene? Why not Modify the Protein? If the gene is modified by site directed mutagenesis then each time the host organism will produce the modified protein. However if the protein is modified each time the protein is produced it has to be modified. Further more chemical modification of protein is: Harsh Nonspecific Has to be repeatedly done

12 12 Directed Mutagenesis A large amount of experimental procedures have been developed for directed mutageneis of cloned genes. All the procedures utilizes : A synthetic oligonucleotide complimentary to the area of the gene of interest but has the desired nucleotide change. What is an oligonucleotide? An oligonucleotide is a short piece of DNA usually 10-30 nt long. A vector e.g. a plasmid or M13. What is M13 ?

13 13 Directed Mutagenesis Directed mutagenesis can be done using : M13 Plasmid DNA PCR Random primers Degenerate primers Nucleotide analogs Error prone PCR DNA shuffling

14 14 Directed Mutagenesis Using M13 For the procedure the following must be known: The nucleotide sequence that encodes the mRNA codon to be changed. The amino acid changes that are to be made. The procedure involves: The gene of interest is inserted into the ds form of the M13 bacteriophage. (M13 has ssDNA and replicated via a dsDNA intermediate). The ssDNA is isolated from the M13 phage.

15 15 Directed Mutagenesis using M13 The ssDNA is mixed with an excess of the synthetic oligonucleotide. The oligo is complimentary to the area of the cloned gene except for the one nucleotide to be changed. The oligo anneals to the ssDNA in the homologous region of the cloned gene. The oligo acts a primer for DNA synthesis using the M13 DNA as a template and the enzyme Klenow fragment of DNA polymerase I. T4 DNA ligase is used to ligate the 2 ends of the newly synthesized DNA. The newly synthesized M13 DNA is transformed into E. coli.

16 16 Directed Mutagenesis Using M13

17 17 Directed Mutagenesis Using M13 Because DNA replicates semi-conservatively half the cells should have the mutant gene. Mutant plaques are identified by DNA hybridization using the oligo as probe. Only 5% of the plaques carry the mutant gene. This makes isolation of those plaques with the mutant gene difficult. To produce large quantities of altered protein, the mutant gene is usually spliced from the M13 DNA by restriction enzymes and cloned into an E. coli plasmid. The procedure has been modified to to enrich for the number of mutant plaques.

18 18 Enrichment for the # of Mutant Plaques One strategy has been to introduce M13 vector carrying the desired gene into an E. coli strain with 2 defective enzymes: A defective form of dUTPase (dut). Cells with defective dUTPase has elevated levels of dUTP which is incorporated into the DNA often replacing dTTP. A defective Uracil N-glycosylase (ung). Uracil N-glycosylase is the enzyme that removes dUTP which is incorporated into DNA during replication.

19 19 Enrichment for the # of Mutant Plaques The procedure involves: The desired gene is cloned into M13 vector. The M13 vector with the desired gene is transformed into E. coli stain dut/ung, which produces ssDNA with 1% of the T replaced by U. An excess of oligonucleotide is added. The synthesis of a second strand occurs.

20 20 Enrichment for the # of Mutant Plaques Addition of T4 ligase. The dsDNA is transformed into E. coli wild type strain. The wild type E. coli with functional ung gene will use Uracil N-glycosylase which will remove the dUTP which was incorporated into the DNA. Therefore the original DNA strand is degraded and only the mutant strand remains. In this way the number of plaques with the mutant gene is greatly increased.

21 21 Enrichment for the # of Mutant Plaques

22 22 Oligonucleotide-Directed Mutagenesis Using Plasmid DNA One of the disadvantages of performing directed mutagenesis using M13 vector is the large number of steps involved. That is: Clone the target gene into M13 vector. Transform into E.coli. Then reclone the gene into an E. coli plasmid. Why are all these steps necessary?

23 23 Oligonucleotide-Directed Mutagenesis Using Plasmid DNA. One approach includes: Inserting the desired gene into the multiple cloning site (mcs) of a plasmid vector. What is multiple cloning site (mcs) of a plasmid vector? Denaturation of the dsDNA of the plasmid by alkaline treatment i.e. dsDNA ssDNA. Why? Addition of 3 distinct oligonucleotide primers: One oligo is designed to alter the target gene. The second is designed to correct a mutation in an Amp resistant gene i.e amp s amp r (SAR) The third oligo is designed to cause a mutation in a tet resistant gene i.e. tet r tet s (RST)

24 24 Oligonucleotide-Directed Mutagenesis Using Plasmid DNA The oligos are added along with 4 dNTPS and DNA polymerase. The oligos anneal and DNA polymerase synthesizes a new strand of DNA. T4 DNA ligase ligates the DNA. The rxn mixture is transformed into E. coli. Transformants are selected for amp r and tet s. How? Using this method >90% of the transformants will have the mutation in the desired gene. The plasmid, E. coli, enzymes and 2 of the oligos are sold in a kit to facilitate wide spread use.

25 25 Oligonucleotide -Directed Mutagenesis Using Plasmid DNA

26 26 Oligonucleotide -Directed Mutagenesis Using Plasmid DNA If we did not have antibiotic markers how could we select for mutant gene?

27 27 PCR-amplified Oligonucleotide Directed Mutagenesis PCR can be used to : §Enrich for the mutant gene §Avoid using M13 vector The procedure involves: The target gene is cloned into an E.coli plasmid. 2 specific oligos are added to the PCR reaction. One primer is complimentary to the target. The other primer is complimentary to the target gene except for the nucleotide that is targeted for change.

28 28 PCR-amplified Oligonucleotide Directed Mutagenesis The oligos maybe overlapping. During PCR the complete target gene and plasmid are amplified. T4 ligase is added to the produce a circularized DNA from the linear PCR-amplified DNA. The recombinant plasmid is transformed into E. coli. Half the cells will have the mutant gene and half will have the wild type gene. The plasmid with the mutant gene can be identified by restriction digestion, PCR or DNA hybridization.

29 29 Directed- Mutagenesis using PCR

30 30 Random Mutagenesis with Degenerate Primers What is a random mutation? So far we have discussed directed mutagenesis at a pre-determined site in a cloned gene. Random mutagenesis involves mutation at any site in the DNA. Random mutagensis is useful because sometimes it is not known which specific nucleotide change that will produce the desired protein. What is a degenerate primer? A degenerate primer is an oligonucleotide where the nucleotides at some positions are varied. ATCCGATGGAATCisoleucine ACCCGATAGAACCThreonine AGCCGATCGAAGCSerine AACCGATTGAAACAsparagine

31 31 Random mutagenesis: Error Prone PCR Some heat stable DNA polymerases used during PCR can occasionally insert the wrong nucleotide generating mutations (Error Prone PCR). By modifying PCR conditions e.g DNA template concentration Adding unequal concentration of each nucleotides Add Mn 2+ It is possible to introduce mutations into the PCR product. This product is then cloned and the modified protein expressed and tested for the desired properties.(3 rd ed only)

32 32 Random Mutagenesis with Degenerate Primers Degenerate primers can be used to introduce random mutations into a target gene. The procedure involves: Insertion of the target gene into a plasmid between two unique restriction sites. Using PCR in separate reactions to amplify overlapping fragments. This requires two pairs of primers (i.e. 4 primers) including 2 degenerate overlapping primers which anneal near the centre of the target gene. Two primers which anneals on opposite strands upstream the unique restriction sites.

33 33 PCR-amplified Oligonucleotide Directed Mutagenesis

34 34 Random Mutagenesis with Degenerate Primers Each reaction has : 1 degenerate primer (2, 4) 1 primer upstream the restriction site (1, 3) After PCR the products are purified and combined. Denaturation and renaturation of the PCR products results in some DNA overlapping the target DNA. DNA polymerase is used to form complete dsDNA. This PCR product is digested with two restriction enzymes for which there are unique sites. The amplified DNA is cloned into a plasmid and transformed into E. coli which will express the modified protein.

35 35 PCR-amplified Oligonucleotide Directed Mutagenesis

36 36 PCR-amplified Oligonucleotide Directed Mutagenesis

37 37 Random Mutagenesis Using Nucleotide Analogs What is a nucleotide? A unit of a nucleic acid consisting of a sugar, a base, and a phosphate. What is a nucleoside? A unit of a nucleic acid consisting of a sugar and a base. What is a nucleotide analog? A nucleotide analog is structurally similar to a nucleotide but is chemically different. E.g. 5 bromouracil is an analog of thymine. A nucleotide analog can be used to cause random mutations in DNA.

38 38 Nucleotide Analog

39 39 Random Mutagenesis Using Nucleotide Analogs The procedure involves: The cloned gene is placed in a plasmid next to two closely placed restriction sites. The recombinant plasmid is treated with the two restriction enzymes to produce 5 and 3 recessed ends and 5 and 3 protruding ends. Recessed is the opposite of protruding, it simply means not sticking out or set back. The enzyme exonuclease III (Exo III) is added and will specifically degrade the DNA from the 3 recessed end only, but not from 5 recessed end or the protruding ends.

40 40 Random Mutagenesis Using Nucleotide Analogs

41 41 Random Mutagenesis Using Nucleotide Analogs After a specific time, the reaction is terminated and the gap produced is filled by Klenow fragment of DNA polymerase I. The dNTP mix used contains 4 normal nucleotides and one nucleotide analog. The nucleotide analog will be incorporated at several places along the DNA. T4 ligase is added to ligate the DNA. The recombinant plasmid with the nucleotide analog is transformed into E. coli. During replication in E. coli the nucleotide analog will direct the incorporation of bases distinct from that in the wild type gene creating random mutations through out the cloned gene.

42 42 Random Mutagenesis Using Nucleotide Analogs

43 43 DNA shuffling Some protein e.g interferons are coded by a family of genes. It is possible to recombine portion of these genes to generate hybrids or chimeric forms with unique properties. This is called DNA shuffling. There are 2 ways of shuffling genes: Using restriction Using DNase1 (deoxynuclease)

44 44 DNA Shuffling with RE Digestion of members of the gene family with RE that cut in similar places. This is followed by ligation of the DNA fragments. This can generate large #s of hybrids which can be tested for unique properties.

45 45 DNA shuffling with DNase 1 Different members of the gene family are fragmented using DNase 1 followed by PCR. During PCR different members of the family are crossed primed. DNA fragments with high homology will anneal to each other. The hybrids generated are then used generate a library of mutants which are tested for unique properties.

46 46 Advantages and Disadvantages of Random Mutagenesis What are some of the advantages of directed mutagenesis? Advantages of random mutagenesis: Many different mutants encoding a wide variety of proteins are generated. Detailed information regarding function of particular amino acids is not necessary. Disadvantages of random mutagenesis : Many mutants have to be assayed to determine which proteins have the desired properties.

47 47 Protein Engineering What did we say protein engineering is? Protein engineering involves the use of genetic manipulations to alter the coding sequence of a (cloned) gene and thus the properties of the protein encoded by that gene. We can use protein engineering to: Improve protein stability Increase protein purity during extraction Increase protein expression Modify cofactor requirement Increase enzyme activity Modify enzyme specificity Study the function of a protein SPECASF

48 48 Improving Stability A variety of enzymes are now used in biotechnology and industry. However many enzymes have limited use because they are denatured on exposure to conditions which are encountered in industrial processes e.g. high temperature, high pH, organic solvents and chemical solvents. What do you understand by protein denaturation? Although thermostable enzymes can be isolated from thermophilic organism, many of these organisms lack the particular enzyme that is required in the industrial process. Gene cloning and site directed mutagenesis has been used to modify enzymes from mesophiles for increased stability.

49 49

50 50 Improving Stability Protein stability can be increased by creating a molecule which will not readily unfold under unfavorable conditions. Protein stability can be improved by: *Adding disulphide bonds *Replacing labile amino acids *Reducing the number of free S-H (sulphydryl) groups.

51 51 Adding Disulphide Bonds Disulphide bonds can significantly stabilize the native structure of proteins. This effect is presumed to be due to the decrease in configuration chain entropy of the unfolded polypeptide. Wild type lysozyme has 2 cysteine residues and no disulphide bonds. Site-directed mutagenesis was used to introduce new cysteine residues and new internal S-S bonds between amino acids: –3 and 979 and 16421 and 142

52 52 Mutagenesis of Lysozyme After mutagenesis each mutant gene was expressed in E. coli. The modified enzymes were purified and tested for enzyme activity and thermostability. The results showed that the thermal stability increased with the presence of disulphide bonds. The most thermostable mutant was the one with 3 S-S bonds. Those mutants which had S-S bonds between amino acids 21 and 142 lost 100% of their activity. Can you guess why?

53 53 Mutagenesis of Lysozyme

54 54 Xylanase Current strategies for the production of paper uses a chemical bleaching step which is essential for the colour and quality of the paper. The bleaching process is used to remove hemicellulose from the pulp. This bleaching agent is a potential toxin effluent. The bleaching process can be enhanced by using the enzyme xylanase. The use of xylanase reduces the amount of chemical bleaching agent and the amount of polluting by- products.

55 55 Xylanase The stage of the process where the enzyme is added is immediately after hot alkaline treatment. In the pulp mills acid is usually added to reduce the pH to near optima of the enzyme. Because of the current trend to reduce the amount of water during processing the pulp remains hot. Therefore a thermostable xylanase is required. One attempt to solve this problem was to produce a modified xylanase (Bacillus circulans) with increase thermal stability.

56 56 Xylanase Site-directed mutagenesis was used to produce 8 mutants xylanase proteins with increase S-S bonds and increase stability. 3 of the mutants were as active as the wild type at 60°C. One mutant with an S-S bond between the C and N terminal ends of the enzyme had twice the activity of the wild type at 60°C. This mutant remained active for 2 hrs while the wild type lost all its activity after 30 min at 60°C.

57 57 Human Pancreatic Ribonuclease Ribonuclease from bull semen (bsRNase) can act as an antitumorigenic agent. The protein is taken up by tumor cells where it degrade rRNA blocking protein synthesis. The dimeric form of the protein is joined by 2 S- H bridges. Antibodies against bsRNase could be produced after prolong use. Therefore human pancreatic RNase (hpRNase) was engineered as an anti-cancer agent

58 58 Human Pancreatic Ribonuclease The aa sequence of bsRNase and hpRNase are 70% identical. The monomeric for hpRNase was modified to form a dimer by changing: Glu 28 Leu Arg 31, 33 Cys Asp 34 Lys When this was expressed in E. coli and solubilized it was a good candidate for an anti- cancer agent.

59 59 Human Pancreatic Ribonuclease Arg Gln Asn Csy Cys Lue Lys Cys Lue A B Soluble, unfolded enzyme Inclusion body Enzyme –glutathione Mixed disuphide Active dimeric enzyme 1. 6 m guanidine HCl 2. Reduced glutathione 1. Dithioretitol 2. Dialysis 1. Dilute 20 fold 2. Reoxidize

60 60 Changing Labile Amino Acids When proteins are exposed to high temperatures deamidation occurs. Deamidation release of NH 3 Asparagine Asparatic acid Glutamine Glutamic acid The loss of the amide groups may result in the lost of activity of the affected enzymes.

61 61 Triose Phosphate Isomerase Triose phosphate isomerase catalyses the interconversion of dihydroxyacetone and phosphate to glyceraldehyde –3 phosphate during glycolysis. The enzyme (Saccharomyces cerevisiae) consist of 2 identical subunits and each subunit has 2 asparagine residues which contributes to its thermal sensitivity. Using oligonucleotide directed mutagenesis: Asn 14 Ile Asn 78 Thr Resulted in enhanced thermostability. When both Asn Asp the resulting protein was unstable even at room temperature.

62 62 Increasing the stability of Triose Phosphate Isomerase

63 63 Reducing the # of Free S-H Groups Interferons interfere with virus replication. They are small protein molecules released from virus infected cells and binds to adjacent cells causing then to produce antiviral proteins which disrupts viral replication. When interferon was cloned and expressed in E. coli it had about 10% of the activity of the authentic form. The E. coli expressed interferon was found to existed as dimers and higher oligomers. Analysis of the DNA of the cloned gene showed that it has 3 cysteine residues which may be involved in intermolecular disulphide bonding resulting in dimers and higher oligomers.

64 64 Interferon It was not know which or if any of the cysteine residues may be involved in intramolecular bonding. A similar molecule interferon have 4 Cys residues at amino acid positions 1, 29, 98 and 138 with S-S bonds between Cys 29 and 138, which is homologous to Cys 31 and 141 of INF.

65 65 Interferon This suggests that Cys 17 of INF was not involved intramolecular S-S bond. Therefore Cys 17 was targeted for mutation to serine. What is the structural relationship between Cys and Ser? Ser has an O atom instead of S atom in Cys therefore cannot form S-S bonds. Sure enough mutation of Cys 17 Ser the resulting INF has specific activity similar to wild type INF. How can INF be use chemotherapeutically?

66 66 Increasing Enzyme Activity In addition to stabilizing the enzyme, site- directed mutagenesis may be used to modify its catalytic activity. To do this detailed geometry of the active site and the amino acids in the active site must be known. Tyrosyl-tRNA synthetase has been modified for increase substrate binding (Km). (If the substrate binding is increased then this increases the rate of the reaction).

67 67 Tyrosyl-tRNA Synthetase Tyrosyl-tRNA synthetase catalyses the the transfer of Tyr to tRNA tyr. This is then added to the growing polypeptide chain. Tyr + ATP Tyr-AMP + Ppi Tyr-AMP + tRNA tyr Tyr-tRNA tyr + AMP The active site of the enzyme was mapped. In the crystal structure of the enzyme, the hydroxyl side chain of Thr 51 form a weak H- bond with AMP of the substrate intermediate of tyrosyl adenylate (Tyr-A).

68 68 Mutagenesis of Tyrosyl-tRNA Synthetase Oligonucleotide mutagenesis was used to create 2 mutations at Thr 51: Thr 51 Ala 51 (removes the H-bond). With this mutation the binding affinity (Km) of enzyme for ATP increase 2 fold. Thr 51 Pro 51. With this mutation ATP is bound 100-fold more tightly.

69 69 Modifying Cofactor Requirement Subtilisins are a class of microbial serine proteases and are widely used as a biodegradable cleaning agents in laundry detergents. Subtilisin binds one or more molecules of Ca 2+ which is important for their stability. Unfortunately subtilisins are used in industrial settings where there are metal-chelating agents which will bind Ca 2+. To circumvent this problem directed mutagenesis was used to abolish the Ca 2+ binding capability of subtilisin and to stabilize the modified enzyme.

70 70 Mutagenesis of Subtilisins The x-ray crystallography structure of the enzyme and the amino acids involved in the Ca 2+ binding was known. Oligonucleotide mutagenesis was used to construct a mutant protein by deleting amino acids 75-83 that is responsible for Ca 2+ binding. The next thing to do was to stabilize the modified protein. aa selected for mutagenesis came from 4 different regions : the N terminus (aa 2-5), omega loop (aa 36- 44), α helical region ( aa 63-85) and a β pleated region (aa 202-222) The mutants were assayed for enzyme activity and stability.

71 71 Mutagenesis of Subtilisins Stabilizing mutations were identified at 7 of the 10 sites. These stabilizing mutations were introduced into a single gene. How could all seven mutations be introduced into a single gene? The results: The mutant subtilisins did not require Ca 2+ as a cofactor. The mutant enzyme was 10 times more stable than the native form in the absence of Ca 2+ and 50% more stable in presence of Ca 2+.

72 72 Increasing Enzyme Specificity Tissue plasmogen activator (tPA) is a protease that is used for the dissolution of blood clot. Treatment with tPA requires an intravenous infusions (1.5-3.0 hrs) because of the clearance of tPA from the circulation is rapid (t½~6 min). For tPA to be effective the patient must be given in high initial concentration which can often cause nonspecific bleeding. Therefore a long life tPA with increase specificity for fibrin in blood clot is desirable. Directed mutagenesis was used to try achieve these goals.

73 73 Mutagenesis of tPA Changing Thr 103 Asn cause tPA to persist in rabbit plasm 10 times longer than the native form ( longer life tPA). Changing amino acids 296-299 from: Lys-His-Arg-Arg Ala-Ala-Ala-Ala produced an enzyme with more fibrin specificity. (LHAA A) Changing Asn 117 Gln causes the enzyme to retain the enzymatic activity of the native form. Combining these three mutations into a single gene allows all three mutations to be expressed in a single protein simultaneously. It remains to be seen if this modified protein will be effective in humans.

74 74 Mutagenesis of tPA

75 75 Streptokinase (Sk) is produced by pathogenic strains of streptococcus and is a blood clot- dissolving protease. Sk complex with plasminogenplasmin degrades fibrin. Plasmin also degrades Sk. For heart attack patients medical personnel has to administer Sk ASAP and in 30-90 min infusions. Therefore a long-lived Sk is necessary. Plasmin cleaves peptide bonds after Lys and Arg residues. Decreasing Protease Sensitivity

76 76 Streptokinase Plasmin cleaves Sk at Lys 59 and 386 and the 328 peptide has only 16% activity as the native molecule. To make Sk less susceptible, Lys at 59 and 386 were changed to Glu by site directed mutagenesis. Glu was chosen to replace Lys because the length of the side chain was similar and Glu does not have a +ve charge. Both single and double mutant retained their activity. Furthermore the half life of all three mutant increase and the double mutant was 21 fold more protease resistant 3 rd ed.

77 77 THE END

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