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BC35C Biotechnology I
(Lecture notes 2004)
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.
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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 3 scheme.
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.
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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.
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Km/Vmax
What is the Km of an enzyme ?
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.
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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.
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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.
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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 ?
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Directed Mutagenesis
Directed mutagenesis can be done using:
M13 Plasmid DNA PCR Random primers Degenerate primers Nucleotide analogs Error prone PCR DNA shuffling
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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. 15
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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 amps ampr (SAR) The third oligo is designed to cause a mutation in a tet resistant gene i.e. tetr tets (RST)
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If we did not have antibiotic markers how could we select for mutant gene?
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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. ATCCGATGGA ATC isoleucine ACCCGATAGA ACC Threonine AGCCGATCGA AGC Serine AACCGATTGA AAC Asparagine
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It is possible to introduce mutations into the PCR product. This product is then cloned and the modified protein 31 expressed and tested for the desired properties.(3rd ed only)
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.
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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.
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Nucleotide Analog
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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.
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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)
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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
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SPECASF
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.
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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.
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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?
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Mutagenesis of Lysozyme
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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 byproducts. 54
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.
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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 60C. 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 60C. This mutant remained active for 2 hrs while the wild type lost all its activity after 30 min at 60C.
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When this was expressed in E. coli and solubilized it was a good candidate for an anticancer agent.
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A
Lue Csy Cys Lys Cys Cys Lue
Enzyme glutathione
Lys
Resulted in enhanced thermostability. When both Asn Asp the resulting protein was unstable even at room temperature.
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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.
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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?
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Tyrosyl-tRNA Synthetase
Tyrosyl-tRNA synthetase catalyses the the transfer of Tyr to tRNAtyr. This is then added to the growing polypeptide chain. Tyr + ATP Tyr-AMP + Ppi Tyr-AMP + tRNAtyr Tyr-tRNAtyr + 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 Hbond with AMP of the substrate intermediate of tyrosyl adenylate (Tyr-A).
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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.
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Mutagenesis of Subtilisins
The x-ray crystallography structure of the enzyme and the amino acids involved in the Ca2+ binding was known. Oligonucleotide mutagenesis was used to construct a mutant protein by deleting amino acids 75-83 that is responsible for Ca2+ 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 3644), helical region ( aa 63-85) and a pleated region (aa 202-222) The mutants were assayed for enzyme activity and stability. 70
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 Ca2+ as a cofactor. The mutant enzyme was 10 times more stable than the native form in the absence of Ca2+ and 50% more stable in presence of Ca2+.
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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.
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Mutagenesis of tPA
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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 3rd ed.
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THE END
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