Site Directed Mutagenesis and Protein Engineering

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Site Directed Mutagenesis and Protein Engineering

• BC35CBiotechnology I(Lecture notes 2004)
• Prepared and presented by
• Dr. Marcia E. Roye
• Office Biotechnology Centre, Ground floor
• Tel [phone] (ext. 2518-20)
• Email marcia.roye_at_uwimona.edu.jm

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Lecture Objectives

• The objectives of these lectures are
• Investigate how desired mutations can be
  introduced into a cloned gene.
• 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), 913670 (1984)
  815662, (1978), 84675.Trends in Biotechnology
  (1990), 816 Biotechnology (1995), 13669,
  Protein Engineering, (1986), 17, 1994, 71379,
  Nature (1989), 342291, Biotechniques, (1987),
  5786, Science (1983) 219666.
• This text and these journal articles are
  available in Dr Royes book rental scheme. 

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Getting notes from Web

• www.uwimona.edu.jm/biochem/courses

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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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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

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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.

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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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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

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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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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.

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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.

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Directed Mutagenesis Using M13
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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.

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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.

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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.

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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.

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Enrichment for the of Mutant Plaques
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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?

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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 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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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 ampr and tets.
  How?
• Using this method gt90 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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Oligonucleotide-Directed Mutagenesis Using
Plasmid DNA
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Oligonucleotide-Directed Mutagenesis Using
Plasmid DNA
If we did not have antibiotic markers how could
we select for mutant gene?
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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.

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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 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.

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Directed-Mutagenesis using PCR
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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.
• ATCCGATGGA ATC isoleucine
• ACCCGATAGA ACC Threonine
• AGCCGATCGA AGC Serine
• AACCGATTGA AAC Asparagine

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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.(3rd ed only)

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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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PCR-amplified Oligonucleotide Directed Mutagenesis
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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.

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PCR-amplified Oligonucleotide Directed Mutagenesis
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PCR-amplified Oligonucleotide Directed Mutagenesis
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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.

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Nucleotide Analog
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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.

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Random Mutagenesis Using Nucleotide Analogs
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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.

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Random Mutagenesis Using Nucleotide Analogs
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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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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.

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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.

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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.

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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
• SPECASF

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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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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 97 9 and 164 21 and 142

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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 by-products.

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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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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

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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.

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Human Pancreatic Ribonuclease
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Changing Labile Amino Acids

• When proteins are exposed to high temperatures
  deamidation occurs.
• Deamidation ? release of NH3
• Asparagine ? Asparatic acid
• Glutamine ? Glutamic acid
• The loss of the amide groups may result in the
  lost of activity of the affected enzymes.

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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.

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Increasing the stability of Triose Phosphate
Isomerase
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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.

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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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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).

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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 H-bond
  with AMP of the substrate intermediate of tyrosyl
  adenylate (Tyr-A).

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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.

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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 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.

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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
  36-44), a helical region ( aa 63-85) and a ß
  pleated region (aa 202-222)
• The mutants were assayed for enzyme activity and
  stability.

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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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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 (t½6 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.

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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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Decreasing Protease Sensitivity

• Streptokinase (Sk) is produced by pathogenic
  strains of streptococcus and is a blood
  clot-dissolving protease.
• Sk complex with plasminogen?plasmin? 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.

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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