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Protein Design and Engineering Topic: Site- directed mutagenesis Presented by: Muhammad wakeel (Roll No:29) Zahid Hussain

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Protein Design and Engineering Topic:

Site-directed mutagenesis Presented by:

Muhammad wakeel (Roll No:29)

Zahid Hussain (Roll No:34)

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Introduction

• Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, either in nature or experimentally by the use of chemicals or radiations.

• Mutagenesis as a science was developed especially by Charlotte Auerbach in the first half of the 20th century.

• There are two major classes of mutation i.e. gene and chromosomal mutations.

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Site-directed mutagenesis

Site-directed mutagenesis, also known as site-specific mutagenesis or oligonucleotide- directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

Site-directed mutagenesis was achieved in 1973 in the laboratory of Charless Weissmann using a nucleotide analogue N4-hydroxycytidine which induces transition of GC to AT.

Michael Smith, 1993 Chemistry Nobel Prize co-winner for developing site-directed mutagenesis

Recently, site-directed mutagenesis has become one of the most commonly used methods in molecular biology.

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PrinciplesIn general, this form of mutagenesis requires that the wild

-type gene sequence be known.

•Thus, enables the mutant oligonucleotides or primers to be synthesized.

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Protein EngineeringProtein 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.

Reference: Zoller MJ. (1991) New molecular biology methods for protein engineering. Curr Opin Biotechnol, 2(4): 526–531. 

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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.

• If the protein is modified each time the protein is produced it has to be modified.

• Further more chemical modification of protein is: Nonspecific Has to be repeatedly done

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Directed Mutagenesis• Directed mutagenesis can be done using: M13 Plasmid DNA PCR Random primer

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Cassette mutagenesis

•In cassette mutagenesis, a synthetic DNA fragment containing the desired mutant sequence is used to replace the corresponding sequence in the wild-type gene.

•It is a simple method for which the efficiency of mutagenesis is close to 100%.

•The disadvantages are the requirement for unique restriction sites flanking the region of interest.

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Cont..The limitation on the realistic number of different oligonucleotide replacements that can be synthesized.•The latter problem can be minimized by the use of doped oligonucleotides.•Doped oligonucleotides usually refers to the use of unequal amounts of each of the four standard dNTPs in oligonucleotide synthesis.

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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.

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Directed Mutagenesis using M13

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• DNA replicates semi-conservatively half the cells should have the mutant gene.

• Mutant plaques are identified by DNA hybridization using the oligonucleotide 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 enrich for the number of mutant plaques.

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Oligonucleotide-Directed Mutagenesis Using Plasmid DNA

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Oligonucleotide-Directed Mutagenesis Using Plasmid DNA.

Inserting the desired gene into MCS.

Denaturation

of the dsDNA i.e. dsDNA ssDNA.

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Oligonucleotide-Directed Mutagenesis Using Plasmid DNA.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.The third oligo is designed to cause a mutation in a tet resistant gene i.e. tetr

tets

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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.Transformation into E. coli.Selection of transformants.>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.

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PCR-amplified Oligonucleotide Directed Mutagenesis

PCR can be used to :Enrich the mutant geneAvoid using M13 vectorThe 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.

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PCR-amplified Oligonucleotide Directed Mutagenesis

The oligos maybe overlapping.During PCR the complete target gene

and plasmid are amplified.T4 ligase is added to produce a

circularized DNA from the linear PCR-amplified DNA.

Transformation into E. coli.50% 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.

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PCR-amplified Oligonucleotide Directed Mutagenesis

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Protein EngineeringProtein engineering is use to alter the coding

sequence of a gene and thus modify the properties of the protein encoded by that gene.

This mutant gene may be expressed in a suitable system to produce unlimited quantities of the modified protein.

Therefore site directed mutagenesis used as a tool in protein engineering to modify the properties of a protein

Reference: Zoller MJ. (1991) New molecular biology methods for protein engineering. Curr Opin Biotechnol, 2(4): 526–531. 

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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 enhanced 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.

Reference: Chiang LW, Kovari et al (2001) Molecular Cloning.

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Xylanase used in paper industryCurrent strategies for the production of

paper uses a chemical bleaching step which is essential for 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.

Ref: Perttula, M., Ratto, M., Konradsclottir, M., Kristjansson, J. K. and Viikari, L., Appl. Microbiol. Biotechnol., 1993, 592–595.

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XylanaseThe stage of the process where the enzyme

is added is immediately after hot alkaline treatment.

Therefore a thermostable xylanase is required.

One attempt to solve this problem was to produce a modified xylanase (Bacillus circulans) with increase thermal stability

Ref: Nissen, A. M., Anker, L., Munk, N. and Lange, N. K., in Xylans and Xylanases(2003).

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Mutant xylanasesSite-directed mutagenesis was used to

produce mutants xylanase proteins with increase S-S bonds and increase stability.

Mutants 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.

Ref: Magnuson, T. S. and Crawford, D. L., Enzyme Microbiol. Technol., 1997, 21, 160–164.

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Human Pancreatic RibonucleaseRibonuclease from bovine seminal

(bsRNase) can act as an antitumor agent.The protein is taken up by tumor cells

where it degrade rRNA thus blocking protein synthesis.

The dimeric form of the protein is joined by 2 S-H bridges.

Therefore dimeric human pancreatic RNase (hpRNase) was engineered as an anti-cancer agent

Ref: Stone SR, Hofsteenge J, Matthies R (1989). "Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily". Biochemistry 28 (25): 9806–9813

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Human Pancreatic RibonucleaseThe amino acid sequence of bsRNase and

hpRNase are 70 % identical.The monomeric form of hpRNase was

modified to form a dimer by changing:Glu 28→ LeuArg 31, 33 →CysAsp 34 → Lys

When this was expressed in E. coli and solubilized it was a good candidate for an anti-cancer agent.

Ref: van der Laan JM, Beintema JJ (1986). "Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases". FEBS Lett. 1992): 338–343

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Modifying Cofactor Requirement of SubtilisinSubtilisin a class of microbial proteases and are

widely used as a biodegradable cleaning agents in laundry detergents.

Subtilisin binds one or more molecules of Ca2+ which is important for their stability.

Unfortunately subtilisins are used in industrial settings where there are metal-chelating agents which will bind Ca2+.

To circumvent this problem directed mutagenesis was used to abolish the Ca2+ binding capability of subtilisin and to stabilize the modified enzyme.

Ref: P. Carter, J.A. Wells, Dissecting the catalytic triad of a serine

protease, Nature 332 (1988) 564–568

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Mutagenesis of SubtilisinsThe x-ray crystallography structure of subtilisin

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 mutants were assayed for enzyme activity

and stability. Ref: A. Volkov, F. Jordan, Evidence for intramolecular processing of subtilisin sequestered on a solid

support, J. Mol. Biol. 262 (1996) 595–599

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Streptokinase• Streptokinase is produced by pathogenic strains of

streptococcus and is a blood clot-dissolving protease and used as a thrombolytic drug.

• Sk complex with plasminogen → plasmin→ degrades fibrin. Plasmin→ also degrades Sk.

• A long-lived Sk is necessary which is resistant to

plasmin.

• Plasmin cleaves peptide bonds after Lys and Arg

residues of Sk. Ref: Young, K Shi, G (1997). "Plasminogen Activation by Streptokinase via a Unique Mechanism.

Texas Heart Institute journal / from the Texas Heart Institute of St. Luke's Episcopal Hospital, Texas Children's Hospital 34 (3): 1–13.

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Mutagenesis of StreptokinasePlasmin cleaves Sk at Lys 59 and 386 and the 328 ,

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.

Furthermore the half life of Sk mutant increases and mutant was 21 fold more protease resistant than native protein.

Ref: Abdelghani TTA, Kunamneni A, Ellaiah P (2005) Isolation and mutagenesis of streptokinase-producing bacteria. Am J Immunology 1(4):125–129

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MCQs(1) Site-directed mutagenesis is used as a tool

fora) Random mutagenesisb) Protein engineeringc) Error-prone PCR2) Protein engineering is use to alter the sequence

of ------and thus modify the properties of the protein encoded by that -------.

a) Geneb) Amino aidsc) Both a & b

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MCQs3) Which of the following properties of the proteins

are changed by Site-directed mutagenesis.a) protein stabilityb) Michaelis constant (Km), V max c) Thermal tolerance, pH stabilityd) Eliminate cofactor requiremente) All of the above4) Thermostability of the enzymes can be enhanced bya) Chemical modificationsb) Disulfide bridges formationc) Replacing liable amino acidsd) Both b & c

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MCQs5) Which of the following enzyme is used in paper

industrya)Proteaseb)Xylanasec)Lipased)Catalase6) -------- a class of microbial proteases which is

widely used as a biodegradable cleaning agents in laundry detergents.

a)Xylanasesb)Subtilisinsc)Pectinases

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MCQs7) Subtilisins used ---------- as its cofactor.a)Ca2+ b)ATPc)Mg2+d)None of the above1)Which of the following acts as a blood clot

dissolving protease.a)Streptokinaseb)Xylanasec)Subtilisind)All of the above

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MQCs1) Streptokinase form a complex with the

followinga) ATPb) Plasminogen, plasminc) None of the above10) Plasmin cleaves peptide bond aftera) Lysine and arginineb) Glycine and alaninec) Proline d) Glutamine

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MCQsWhich of the following method is of random mutagenesis?a.M13 mutagenesisb.PCR amplified mutagenesisc.Primer extension mutagenesisd.DNA shufflingGene of interest is inserted into M13 vector in:a.ss DNAb.ds DNAc.both

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Random mutagenesis by degenerate primers involves:a.4 different primersb.pairs of 2 different primersc.Only 1 pair of primer d.Only degenerate primersIn Mutagenesis by M13 plasmid enzyme used for synthesis of DNA is :a.DNA polymerase b.RNA polymerasec.Klenow fragment of Polymerase d.Both a & c

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5-Bromouracil is analogue of a.Thymineb.Guaninec.Cytosined.Uracil3 oligonucleotides are used in directed mutagenesis bya.M13 phageb.PCRc.Plasmid DNAd.All of the above