08 engr prot therapy032911

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Lecture 8: What additional tools are

available from, and what are theapplications of, the products of molecular

biotechnology. How are they important

to human health, quality of life and

society?

Directed Mutagenesis (Chapt 8: pp290-329)Diversity and biodiversityProtein Engineering (Chapt 8: pp290-329)Therapeutic Agents (Chapt 10: pp379-399)


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http://en.wikipedia.org/wiki/Epoetin

simple protein molecule[proteins are the workhorse of the cell and body]

Goal is to mimic Nature, and use!


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http://en.wikipedia.org/wiki/Epoetin

simple protein molecule[proteins are the workhorse of the cell and body]

Goal is to mimic Nature, and use!

and improve


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Mutagenesis

In vivoIn vitro


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Mutagenesis

In vivoIn vitro

Two thoughts:Mutations occur spontaneously

Single base changes in the genomeIndels Duplications

Mutations occur in response to stimulusSingle base changes in the genomeIndelsDuplications

Mimic or expedite naturally occurring mutationsAttractive given recombinant DNA technologyOverproduction and purificationMake better version

What is the basis for changing parts of the genome? Cell? Individual?


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Natural selection and growth: How?

[bacteria as a model]

Source of biodiversityMutations and Natural Selection/ EvolutionBiological perspective of mutations: resource limitation

Glucose- depletedFructose- not availableMaltose- not availableLactose- available


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or in the presence of

selection event

Population overgrowth -> colicin[Antibiotic challenge -> ampicillin]

George Church, @ISB symposium 10/10/08 natural and prevalent antibiotic resistanceEnvironmental challenge -> radiation, heavy metals, poisonsNatural selection vs adaptive evolutionWhole organism, population versus molecular level


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Bacterial modeling 1

Random neutral mutations vs adaptive evolutionRLenski (MSU) long-term evolution experiment1998: 12 strains E. coliFeb 2011: 50,000 generations, sampled at every 500SJGould: replay lifes tape (fossil record)RWoods and JBarrick: slow and steady often win evolutionary race; hares might eventually lose

hidden potential mutations with no obvious advantagesGenetic interactions that reduce benefit of certain regulatory mutations in losers-> outcompetedWoods, Barrick, Cooper, et al. (Mar 11) Sci 331:1433

http://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benefits-of-being-slow-and-steady/


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SJGould: replay lifes tape (fossil record)Replay: 10 clones from each of four strains at 500 gen +900 genVertical axis is measure of evolution, ie change from original

@1500 generation- many had two mutations (some dating to 500th gen)- winnersBoth winners and losers were more competitive than 0th gen.losers were better adapted than winners in competition after 350 gen

Winners accumulated mutations ~100 gen earlier than losersQuicker and more dramaticallyAt 800 gen, made up for losers headstart and can outcompete

Not faster evolution, eg same number of mutationsWinners had mutation in spoT [affects DNA packaging] [ppGpp hydrolase]

Causes global genome changes, on/offLosers had mutation in topA [controls packaging]

Negates spoT advantages and suppresses spoT mutationshttp://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benefits-of-being-slow-and-steady/


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SJGould: replay lifes tape (fossil record)Replay: 10 clones from each of four strains at 500 gen; +900 genVertical axis is measure of evolution, ie change from original

@1500 generation- many had two mutations (some dating to 500th gen)- winnersBoth winners and losers were more competitive than 0th gen.losers were better adapted than winners in competition after 350 gen

Winners accumulated mutations ~100 gen earlier than losers

Quicker and more dramaticallyAt 800 gen, made up for losers headstart and can outcompete

Not faster evolution, eg same number of mutationsWinners had mutation in spoT [affects DNA packaging] [ppGpp hydrolase]

Causes global genome changes, on/offLosers had mutation in topA [controls packaging]

Negates spoT advantages and suppresses spoT mutationshttp://blogs.discovermagazine.com/notrocketscience/2011/03/17/replaying-evolution-reveals-the-benefits-of-being-slow-and-steady/


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Role of history in evolutionSJGould: innocuous historical events can have massive repercussions, often the differenceBetween survival and extinction every genetic change is an accident of historyEvolution is fundamentally quirky and unpredictable (Wonderful Life)Lifes tape replayed- evolution could go down very different pathsSCMorris: Any number of evolutionary routes, but destinations are limitedSee convergence on same adaptations-> history has little effect on evolutionLifes tape replayed- evolution gives the same results, more or lessGrow in low sugar so is limited after a period of timeCitrate present but cannot be used if oxygen is presentIn 20 yrs of evolution, only 1 clone has done so

http://blogs.discovermagazine.com/notrocketscience/2008/06/02/history-restricts-and-guides-the-evolution-of-innovations/


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Role of history in evolutionSJGould: innocuous historical events can have massive repercussions, often the differenceBetween survival and extinction every genetic change is an accident of historyEvolution is fundamentally quirky and unpredictable (Wonderful Life)Lifes tape replayed- evolution could go down very different pathsSCMorris: Any number of evolutionary routes, but destinations are limitedSee convergence on same adaptations-> history has little effect on evolutionLifes tape replayed- evolution gives the same results, more or lessGrow in low sugar so is limited after a period of timeCitrate present but cannot be used if oxygen is presentIn 20 yrs of evolution, only 1 clone has done soTwo explanations1) Result of random mutation2) Function of the particular history of the population


prediction


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Two explanations1) Result of random mutation

If so, could come up at any generation2) Function of the particular history of the populationIf so, appears in later generations, function of earlier mutationThree replays, sampling from points in the pastCitrate users evolved much more often in later generations than earlier onesCitrate use is not simple due to extremely rare mutation (1 in 10 trillion)First mutants were not efficient at using citrateAdditional mutations neededDivided population into sugar specialist and sugar+citrate generalist

Blount, Borland, Lenski (08). PNAS 105:7899


Experimental data


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Molecular evolution

MKimura, 1968: Neutral Theory of Molecular EvolutionOriginally thought of as argument against Darwins theory of evolution by natural selectionMaintained, most agree, two theories are compatible

But provides a large role to genetic driftNeutral: differences to not influence fitness of individual

1) Based on degenerate genetic codeSynonymous vs Non-synonymous mutationsGCC and GCA => Ala

2) Also based on majority of AAc changes being neutral

http://en.wikipedia.org/wiki/Neutral_theory_of_molecular_evolution


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Evolution theory

1972 NEldredge and SJGould. Paleobiology 3:115: Punctuated EquilibriumDegree of gradualism (commonly attributed to Darwin)

is virtually nonexistent in the fossil record stasis dominates the history of most fossil speciesMisconceptions:1) species originate more or less overnight, in a single step

rapidly is in the context of a geologist2) species undergo no further evolutionary change once speciation is complete

is actually a form of gradualismRDawkins: apparent gaps represented in fossil record document migratory events rather than

evolutionary events; evolution certainly occurred but probably gradually elsewherePE is a minor gloss, an interesting but minor wrinkle

Non-scientists: micro vs macro/evolutionEHDavidson: role of gene regulation (gene regulatory networks) in evolution; genome of sea urchin

http://en.wikipedia.org/wiki/Punctuated_equilibrium


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Natural mutation: HBB, beta hemoglobin gene

Molecular, genetic and biochemical perspectives1600 bp, three exonsmRNA 626 bp; 44 bp codes for AAc

Normal rbc ~120d; sickle cell ~10-20d

Left: hemoglobin, green and blue= alpha chainsGold and aqua= beta chainsGold spheres= phosphates; box= Glu6Variant at 6= most common variant sickle cell HBS

Right: clumping of two hemoglobins with variant AAcSeveral 100 HBB variantsAutosomal recessive

http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/hbb.shtml


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Spontaneous chemical reactions

Naturally occurring mutationsEnergy driven, eg UV light (tanning)Chemical basis of mutations

Bases undergo random modificationsIf uncorrected, mutation is hard copiedSNP


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Another perspective: people and mutations

Essay, NYTimes. Oct 9, 2007. BHLerner, MDLongevity reflects how the understanding of sickle cell disease has changedInitially misdiagnosed in early 1960s- found its way into the public consciousness.

First complaints of joint aches- met with skepticismAt 16, diagnosed with both sickle cell anemia and beta-thalassemia

Combined condition may be less severe than pure sickle cell disease, contribute to longevity[?]Course of disease: painful sickle cell crises, spleen removal, shoulder surgery, degeneration of hips

Fear of promoting drug addiction: under-prescribed pain meds, requiring blood tests first


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Site-directed mutagenesis

Theoretically, can modify protein at either the protein-level or the gene-level@Protein-level: chemical, post-translational, modifications are harsh, nonspecific and tedious

But, mirrors Post-translational and epigenomic@Gene-level: Structure and predictive tools necessary

~easierDirected mutagenesis -generate amino acid coding changes at DNA level


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Evolution of genetic engineering(playing god[s])

ExperienceBread, beer, agriculture and naturally occurring products and processes

[Modern] ways and means, biotechnologyBiology, genetics

[Ultra-modern] ways and means, biotechnologyRecombinant DNA technology, genomics, bioinformatics, funding

Improve upon Natureout of context

More and inexpensive supply[cadavers vs carcasses vs controlled products and processes]

Adaptation[extremophile products and processes]Genetic engineering

Directed mutagenesisProtein engineering


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Limited, now, by imagination and creativity

Recombinant DNA technologyBacillus stearothermophilus, 90oC hot springs, -amylase

At high industrial-scale temperatures, converts starch to alcoholNaturally occurring product or process not optimized for use

Small-scale to industrial scaleYields, efficiencies, stability

Directed evolution: Why?Km and Vmax (substrate binding and catalytic efficiency)Thermal tolerance, pH stability (structure and function)Activity in nonaqueous solvents (enhanced nonphysiological)Remove or alter cofactor needsLink enzymes/proteins/subunits togetherAlter substrate-binding site and other binding/recognition sitesAlter resistance to proteases, extending half-life

Alter allosteric regulationAlter trafficking and addressing, eg secretion/segregation/placementTracking or visualizing proteinsPurification issues

Directed evolution: How?Mimic natural mutationsAlter amino acids as directed mutagenesis


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Oligonucleotide-directed mutagenesis[Example of imagination and creativity]

Biology and geneticsssDNA M-13; E. coli plasmids; DNA replication and mutagenesis

Protein chemistry and biochemistryStructure/function

Recombinant DNA technology [and synthetic chemistry]Alter nucleotide sequence to alter amino acid sequence to alter structure/function

Results: 50-50 recovery of WT vs mutant


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v2: Enrichment of mutated gene

Another approach:RF with cloned WT gene, grown in dut ung strain

[incorporates and leaves U]Add oligo with mismatch nucleotide

- strand synthesized with UTf WT hostWT gene sequence is removed by repair mechanism

Results: 50-50 recovery of WT vs mutant (in theory)[[we dont know everything about Nature:]]Only 1% of mutants recovered

Need to enrich


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Mismatch repair: simple

Products and Processesas biotechnological applications

DNA replicationDNA repair

dut/ungMismatch repair

Beneficial to biotechnological applicationsDeleterious to biotechnological applications


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v3: Bypass multiple steps

Another approach [Direct]:Plasmid (ALTER) with cloned WT gene

Original ApS and TcRAdd three mismatched oligos:

Mutation + ApR and TcSAdd T4 DNA polymerase and T4 DNA ligase

Results: mutated gene with easy selection[Repair mechanism of E. coli]

[[using a process as biotechnology]]

Time-consuming multiple stepsMake RF form with WT geneGo through ssDNA formRecover mutated form as dsClone into plasmid to use


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v4: Applying newer technology to simplify: PCR

Simpler and faster, and more versatile [more possibilities]PCR to introduce mutation and enrich for mutated geneFour types of mutations possible:

Point mutationDeletion mutationInsertion mutation

LargeSmall


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v5: Applying newer processes to maximize:Error-prone PCR mutagenesis

Recall the basis for evolution (and natural selection)Mechanism of molecular evolution

More versatile [more possibilities]PCR to introduce multiple, random mutationsTaq pol inherently error-prone, lacks proofreading activity

From 1 to 20 errors in 10kb[but 1-2 per 1,000 is limited number; usually one mutation per triplet codon]

Natural selection and subsequent artificial selectionReplication biochemistry- enhancing errors

Mn++ for Mg++; nucleotide pools; othersSuccess: enzymes with improved solvent and temperature stability; specific activity


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v6: Applying newer inexpensive technology to optimize:Degenerate oligonucleotide primers

Simpler and faster, and more versatile [more possibilities] AND semi-controlledDNA [Replication/Repair] Polymerization to introduce multiple, random mutations

At G position/vial, additional nucleotides provide random [mis]incorporation at G


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v6: Applying newer inexpensive technology to optimize:Degenerate oligonucleotide primers

Simpler and faster, and more versatile [more possibilities] AND semi-controlledDNA [Replication/Repair] Polymerization to introduce multiple, random mutations

At G position/vial, additional nucleotides provide random incorporation at GPCR primers used to synthesize novel geneRE digests to cut out geneNatural selection and subsequent artificial selection


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v7: Random insertion/deletion

Limitations of PCR error-prone mutagenesisAlternative technique:

delete small number of nucleotides at random positions and insert specific or random sequences into that position

Gene with RI-H3 ends [or any two unique]Add non-P linker to one [[no P= no ligation, so gapped]]Close circular DNA with T4 ligase and

repair degraded with T4Pol, extending nick to remove strandProduces ss, with red insertionRandom nick ss with Cerium[IV]-EDTA complexLigate with RE-linker plus additional nucleotidesPCR amplifyRemove linkers with REBlunt end-filling and close/ligate into plasmidTest for activity


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Naturally occurring mutations and evolution, eg -interferonRelated genes with slightly different biological activity

Make hybrids containing parts or these genesGoal is unique properties, eg, combine attributes of two or more originals

High enzymatic activity plus thermostability

v8: DNA shuffling: creating hybrids or chimeras


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v9a: DNA shuffling: naturally occurring RE sites

Simplest solution:Use RE fragments to generate hybrid

2 genes, ea with same 3 unique RE sites14 hybrids


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v9b: DNA shuffling: random fragmenting

Alternative solution:Use fragment several genes randomly with DNaseISelect small fragmentsPCR fill and amplify [will cross-amplify if hybridize]Finish with terminal primers for ends

Can do this protocol with genes from different families, with limited homologyCan do variation protocol with genes from different families, with no homology


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Can do variation of protocol with genes from different families, with no homologyNot PCR-basedSeveral DNA fragments combined and partially digested with DNaseI

Blunt filled with T4 DNA polSize fractionateAdd synthetic DNA fragment forming hairpin loop with specific RE

To prevent long concatenationsRE digest off hairpin endsPut into vector

v9c: DNA shuffling: nonhomologous random fragmenting


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Add non-standard amino acid with unique R groupAmino acids are modified post-translationally in eukaryotes

Different chemical and biochemical propertiesexample, OH-Pro, major component in collagen

Engineer E. coli host with novel tRNAex, tyr-tRNA synthetase from Methanococcus jannaschiiAdds novel amino acid at amber codon, UAG normally stop

O-methyl-L-Tyrex 2, aminobutyrate using Val-tRNA synthetase

Inserting non-standard (unusual amino acids) into mutant


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Protein engineering

1000s of enzymes studies and biochemically characterized

~20 account for >90% used industrially

Why?naturally-occurring enzyme is optimized for a niche

Not suited for highly specialized industrial applicationex, high temperature stability, organic solvents (many industrial processes)

Thermophilic organisms may not have particular desired catalytic function


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Altering thermostability

Structure ~ FunctionNot just thermostability

Often resistant to denaturation by organic solvents and nonphysiological conditions- ex, pHAdding disulfide bonds can increase stabilityProblem: how do these perturb the native structure?


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Altering thermostability: example, T4 lysozyme

Oligonucleotide-directed mutagenesis to increase structural stability at higher temperaturesSix variants with new di-S bonds

2, 4 or 6 AAc changes to Cys at the same timePositions- spatially close to each other in the active enzyme structure

But not in active siteGenerate 1, 2 or 3 di-S bonds

Activity not predictable, eg, 2 additional S-S in one case gives activity at higher temperature


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Altering thermostability: example, xylanaseUse of computer modeling and crystallographic data

Paper making processWood pulp chemically treated [bleaching] to remove hemicellulose,

contributes to discoloration of paper productCreates large amount of toxic effluent

Alternative is using enzyme: Bacillus circulans xylanaseBut, follows hot alkali stepComputer modeling to determine placement of 1, 2 or 3 di-S bonds

All 8 mutants showed increase in thermostability, relative to WT3 were as active as WT at 60oC1 was twice as active at 60oC and retains 85% activity after 2 hr at 60oC

WT is unstable after 30 min


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Adding di-S bonds to produce new application: example, RNase

RNase or ribonuclease from bull semen can act as an anti-tumorigenic agentin vivo and in vitro, dimeric form is internalized into tumor cells by

non-receptor mediated endocytosisSelectively degrades rRNA in cytoplasm; Blocks protein synthesis --> Cell deathAnti-tumor activity due to dimer structure

Of all pancreatic-like RNase superfamily members, dimer structure only found in bull semenBut, clinical application generates human antibodies to bull semen RNase

Does not allow multiple applications or prolonged useEngineer human pancreatic RNase to be a dimer [70% identical] to function as anti-tumor agent


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Adding di-S bonds to produce new application: example, RNase

[mammal cells are not all alike]

Engineer human pancreatic RNase to be a dimer [70% identical] to function as anti-tumor agentDimeric human RNase insoluble, segregated into inclusion body


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Protein engineering to increase stability: temperature sensitive amino acids

Asn and Gln at high temperatures undergo deamidation toAsp and Glu, different chemical and biochemical properties

structure ~ function changesExample, yeast triosephosphate isomerase

Homodimer with 2 Asn per monomer, located at protein-protein interfaceChanging eitherto Thr or Ile enhanced thermostabilityChanging eitherto Asp decreased thermostability, as predictedChanging both to Asp decreased stability at normal temperature and lowers activity

Another example, long-acting human insulin: Gly for Asp increased half-lifeRecently approved for use as human therapeutic agent


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Protein engineering to increase stability: Are too many di-S bonds bad?

Expressed foreign protein maybe less active than expectedHuman IFN- cloned and expressed in E. coli

10% antiviral activity of native glycosylated formYields good; Most of product as dimer or multimer --> inactive

Native has three Cys residuesPossible di-S bonds in E. coli forming multimers, but not in humanChange to Ser ->similar, but OH vs SH

Reasoned Cys-17 not involved in di-S bond in human, so changed it to SerSer-17 form has specific activity similar to native and more stable during long-term storage


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Protein engineering to increase enzymatic activity

Given 3D structural information, including active site dataIncrease activity by modifying substrate-binding specificityPredictions of changing amino acids

Thr-51Replace with Ala or Pro

OH of Thr-51 forms long H-bond with tyrosine adenylateRemoval of weak H-bond may improve affinity of enzyme for ATP

Tyr + ATP -> Tyr-A +PPiTyr-A + tRNATyr + AMP

Tyrosyl-tRNA synthetase (B. sterothermophilus)

Theoretically, Pro distorts -helixShould not work


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Protein engineering: new enzymatic activity

Theory: mutagenesis plus select for new desired activity and against old activityExample: endoprotease

Cleaves between adjacent Arg residuesError-prone PCR; fuse to E. coli surface protein [blue], with negative chargesTwo different substrates used to score selection

1) 3x Arg with 2 fluorescent dyes, at ends of protein, with positive charges2) 3x Arg with 1 dye, at one end of protein, with positive charges

Select for (1) and against (2) using fluorescent cell sorter1 clone found with >3Mx selectivity for new Ala-Arg over old Arg-Arg activity


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Protein engineering: modifying metal cofactor requirements

Subtilisins, Ser proteases secreted into mediaWidely used as bidegradable cleaning agent in laundry detergents (B. amyloliquefaciens)

Binds tightly to Ca++ which stabilizes enzymeIndustrial protocols include chelating agents

Strategy to remove Ca++ binding, then to increase stability of modified enzymeCrystallographic data: residues 75-83 binds Ca++ --> delete but retains overall structureRandom mutagenesis: residues that interacted with 75-83 as targets --> 10 residuesSeveral rounds of stabilizing mutations but low levels of activityCombine mutations for stabilized mutant without Ca++ requirement and high level of activity


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Protein engineering: modifying metal cofactor requirements

Targets: N-term (2-5); omega loop (36-44); -helical region (63-85); -pleated region (202-220)Mutate; grow and heat to 65oC/1 hr; assay for subtilisin activity

Use B. subtilis, product kills E. coliAfter initial screening, 7 stabilizing mutations out of 10 positions targetedSecond round, combine mutations into one gene

Mutant is 10x more stable than native in absence of Ca++ and 50% more stable in presence of Ca++-> complex properties involving large number of amino acids can be engineered


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Decreasing protein sensitivity

Streptococcus streptokinase, 47 kDa protein that dissolves blood clotsComplexes with plasminogen to convert to plasmin, which degrades fibrin in clots

Plasmin also degrades streptokinase [feedback loop]In practice, need to administer streptokinase as a 30-90 min infusion [heart attacks]A long-lived streptokinase may be administered as a single injection

www-s.med.uiuc.edu; JMorrissey: Med Biochem 10/30/06 http://sandwalk.blogspot.com/2007/04/blood-clotting-basics.html


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Protein engineering: decreasing protease sensitivity

Streptokinase (Streptococcus bacteria) is used as a blood clot-dissolving agentForms complex with plasminogen to convert plasminogen to plasmin

Plasmin degrades fibrin in clots

Plasmin degrades streptokinase [[example of feedback loop]]

Has to be administered as a 30 to 90-min infusion after heart attackDesire single injection and rapid transportation to hospital

Plasmin cleaves at Lys-59 and also at Lys-386Strategy: Lys (green) to Glu (red)

Similar side chain and not charged to retain 3D structureBoth single mutants plus double mutant have activities of nativeHalf-life in presence of plasmin increased, with double mutant ~21x more protease resistant


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Protein engineering: modifying enzymatic activity,eg, protein specificity through recognition

Catalytic activity includes recognition of specific substrate and subsequent action>2,500 RE discovered

Many recognize same sequence -> 200 different recognition sitesMajority recognize 4-6bp sequences, not useful for generating large fragments like 8+bp cuttersFokI endonuclease, relatively nonspecific (Flavobacterium okeanokoites)

Second type of DNA-binding proteins: Zn++ finger proteins, eg murine Zif268 with 3 fingersBinds DNA with finger interacting with specific 3 nucleotides

Recombinant protein contains His tag to aid in protein purificationUnder control of T7 promoter (expression cuts host DNA)

Results: two hybrid FokI REs that cut lambda DNA#1 is specific at target site#2 also cuts two other sites

Zn++ finger proteins recognize 3 bases but may also recognize 2 of 3Promising [[Proof of concept]]


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Protein engineering: modifying binding activity,eg, protein specificity [non-enzymatic]

AntibodiesBinds antigensMostly same structure except hypervariable region

Unique and highly specific for antigenic determinantRegion called Fab fragment

http://en.wikipedia.org/wiki/Antibody


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AntibodiesMostly same structure except hypervariable region

Unique and highly specific for antigenic determinant

Region called Fab fragmentBinds in absence of rest of AbTwo peptide chains: heavy and light

each with different hypervariable complementarity-determining regions (CDRs)each with similar framework regions (FRs)Modify CDRs to change binding/recognition specificity

As a dimer, complications for modification ->


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As a dimer, complications for modification ->6 CDRs total in two chains; modifying one or more amino acids changes specificityRandom mutagenesis PCR to mutate 3 CDRs of heavy-chain gene separatelyCombine into single peptide

Example, Fab of monoclonal Ab specific for 11-deoxycortisol(precursor to cortisol [stress hormone] and hypertensive)

New version recognized cortisol and not 11-deoxycortisolPromising [[Proof of concept]]: screen library of mutagenized Fab genes for any antigenic determinant


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tPA, tissue plasminogen activatorMultidomain Ser proteaseDissolves blood clots [like streptokinase]Cleared rapidly from body, so needs to be infused

Requires high initial concentration to be effectiveSide effect of causing nonspecific internal bleeding

Desire: long half-life; increased specificity for fibrin; decreased nonspecificMethod: directed mutagenesisResults:

Thr-103 to Asn -> 10x longer life in rabbit plasma;Lys-His-Arg-Arg (296-299) to Ala-Ala-Ala-ala -> more specific for fibrinAsn-117 to Gln -> fibrinolytic activity as native

All three into one mutant[2010]: testing to validate as substitute for native tPA

Protein engineering: increasing enzyme stability and specificity


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Fructosyl-amino acid oxidaseGlycation- nonenzymatic addition of glucosyl residues on surfaces of blood proteins

ex, hemoglobin, albuminIncreases in diabetics with high blood glucose levels

Does not follow food intakeMay be a good way to monitor diabetes patients during therapy

ex, hemoglobin A1c (HbA1c) level measures Val glycationCan use Fructosyl-amino acid oxidase (Corynebacterium sp)

Specific for D-fructosyl-L-Val on hemoglobin;No activity for N-fructosyl-L-Lys on albuminBut Fructosyl-amino acid oxidase (Corynebacterium sp) enzyme is unstable

Clone into E. coli and repeated rounds of in vivo mutagenesis/screening for stability 47oC/10 minsimple directed evolution in four rounds

Protein engineering: increasing enzyme stability II[Application and example of evolution and selection theory]


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Enteropeptidase (or enterokinase), membrane-bound Ser proteaseFrom duodenal mucosa; two polypeptide chains converting trypsinogen to trypsin

Bovine or porcine versions used to excise poly-His tags from recombinant proteins from E. coliBut also cleaves other sites too- lowers yield/modifies recombinant proteins [[nonspecific]]

Difference between lab-scale vs industrial-scale (worry more aboutyields)Alternatives: medaka- freshwater fish (Oryzias latipes)

Comparable enzymatic activity, same recognition site10% activity against similar [nonspecific secondary] sites vs bovine/porcine versions

Study medaka enzyme to understand basis for strict specificity requirementsLook for amino acids conserved in four different mammalian enzymes not in medaka

5 sitesMutate each in medaka to reflect mammalian version, see 1 with less nonspecific activity

Generates enzyme with altered specificity and more biotechnological applications

Protein engineering: Changing enzyme specificity III


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Generally, directed mutagenesis addresses 1 property at a time1 amino acid change may alter structure ~ function, other parameters

Requires compensating mutationsLabor-intensive and tedious; alternatives-Molecular breeding: novel protein from set of existing similar proteins

Does not require prior knowledge of structure/functionProof of Concept expt:

26 different subtilisin genes from different Bacillus strainsDNA shuffle to produce library of chimearsSubset of 654 clones into B. subtilisAssay secreted proteins, plus parentals

23oC activity; thermostability, solvent stability, pH dependence[most useful in industrial applications]

Natural selection rarely selects for optimal activity at 23oC activity and 70oCResults: 77 as well as or better at 23oC activity

Sequenced-> all were chimeric1 had 8 crossovers with 15 amino acid substitutions

Protein engineering: Changing multiple properties simultaneously


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First to combine two site-directed mutagenesis techniques with gene shuffling and sorting proceduresDirected evolutionJCherry at Novo Nordisk Biotech/Davis, CA. deliberate and random mutations can be screened for a commercial product..

-Maxygen Inc/Redwood City, CA[Broad Institute: Coprinus cinereus 37.5 Mb genome sequenced]

Laundry, detergent and mushrooms

http://www.wildaboutbritain.co.uk/gallery/g; http://www.education.umd.edu/EDMS/mislevy/Drawings/washing.jpe; http://www.fotosearch.com


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Generally, directed mutagenesis addresses 1 property at a time1 amino acid change may alter structure ~ function, other parameters

Requires compensating mutationsLabor-intensive and tedious; alternatives-

#1 DNA shuffling to isolate hybrids- need two or more similar genes

#2 Random mutagenesis or error-prone PCR- essentially make two or more similar genes

Combine #1 with 1 of #2Biotechnological application: Peroxidase

Ink cap mushroom Coprinus cinereusDye transfer inhibitor in laundry detergentOxidizes [decolorizes] leached dues and

prevents re-staining other clothesNeed high pH, temperature and peroxide levels

http://en.wikipedia.org/wiki/Coprinopsis_atramentariahttp://www.nature.com/nbt/press_release/nbt0499.html


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Biotechnological application: PeroxidaseInk cap mushroom Coprinus cinereusDye transfer inhibitor in laundry detergentOxidizes [decolorizes] leached dues and prevents re-staining other clothes

Need high pH, temperature and peroxide levelsMethods:

Site-directed mutagenesis to replace solvent-exposed amino acids with nonoxidizable side changes

Introduce stabilizing features, eg, di-S bridgesError-prone PCR to identify other beneficial mutations

Results: successful mutations combined into 1 hybrid- 114x thermal stable; 2.8x oxidative stable

But, not optimal under actual wash conditionsAdditional DNA shuffling: 174x thermal stable; 100x oxidative stableUsed as dye transfer inhibitor in laundry detergent

Also, model for other enzyme development[SJKimBKSong (Mar10) Biocatalysts and Bioreactor Design][CherryPedersen (99) Nat Biotech

Directed evolution of a fungal peroxidase]


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Therapeutic agents

Human health, quality of life and societyPrior to recombinant DNA technology, most human protein pharmaceuticals were available

in limited quantities [cadavers, carcasses]Costly to produce, modes of action not well-characterized

HIV, Hep-B from blood-derived products (hemophiliacs)Evolution of therapeutic agents

Natural productsAccidental discovery/use of mixtures to isolation/use to synthesis by Nature to Organic Chemistry (Age of Industrialization) to proteins (and antibodies) to

Derived and modified natural productsrecombinant DNA technology (Molecular cloning/Protein engineering) to

Back to natural products (Bioprospecting)

Enhanced understanding and identification/characterization/development for useDiversity

BiodiversityCulture diversity


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Protein therapeutic agents


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Protein therapeutic agentsPrior to recombinant DNA technology

Most human protein pharmaceuticals were available in limited quantities, Extremely costly to produce and biology not well-characterized

Animal sourcesHorse/cow sera- antibodies; influenza vaccinePig insulin prior to 1982

Blood donors- blood, blood components (clotting agents/hemophilia)Cadavers- human growth hormone from pituitary glands; blood clot factors

Recombinant DNA technologySufficient quantities for efficacy testing and human useSeveral thousand different proteins clones (2009)

500 undergoing clinical testing250 biotech drugs approved in US and/or EU

2006, estimated annual global market for recomb protein drugs at $60B10 blockbuster drugs accounted for 50% of this

Rituxan (rituximab)- monoclonal Ab for non-Hodgkin lymphoma @$4BRecombinant insulin @$2.5B

1985: Genentech- FDA approval to sell first biotech industry product, recombinant human growth hormone

Hepatitis B vaccineRecombinant human insulin


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Pharmaceuticals: proteins, cDNAs and therapeutics

General strategies for obtaining cDNA (clone)- early eraPurify protein; determine partial amino acid sequence; make oligo and search librariesPurify protein; make antibody against protein; screen libraries

Considerations include quantity of natural state protein and locationex, Insulin is major protein synthesized by islets of Langerhans of the pancreas;

70% of its mRNAPrior to whole human genome sequencing, difficult to isolate low copy or uncharacterized genes

Human interferon proteins: , , - each with different biological activityGenomics, bioinformatics, systems biology, technology


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Pharmaceuticals: interferon cDNAs

Human interferon proteins: , , - each with different biological activity1980s IFN thought to be 1 proteinSite of synthesis unknown; low concentration

E. coli eukaryotic protein expression vectors not readily available


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Interferon cDNAs: genomics and bioinformatics,and medicine

Human interferon proteins: , , and synthesized in cells that have been exposed to viruses or viral RNA synthesized in response to cell growth-stimulating agents

coded by 13 different but similar genes; encoded by 2; by 1

Antiviral activities vary

1 and 2 are similar when challenged with a virus-challenged bovine cell2 is 7x more effective than 1 when human cells are treated with virus2 is 30x less effective than 1 when mouse cells are used in this assay


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Interferon cDNAs: genetic engineering

Gene shuffling to obtain hybrid protein with novel properties1 and 2 as parentals, use REs to shuffle or PCR-basedHybrids expressed in E. coli

Test for extent of protection against viruses; antiproliferative activity against various human cancersSome hybrids have passed clinical trials and approved for use as human therapeutic agents


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Interferon cDNAs, optimization

Some hybrids have passed clinical trials and approved for use as human therapeutic agentsLonger-acting interferon- minimizes side effects, lower dosage, lower frequency of treatment

Method: PEG (polymers)Method: fuse with a stable protein, eg, human albuminResults: native is removed after 2 days; hybrid effective for two weeks

Hepatitis C (Phase III, 2006)


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Pharmaceuticals: human growth hormoneHGH or somatotropin is a 191 amino acid pituitary hormone, MW 22,125 daltons

Stimulates production of insulin-like growth factor 1Children: controls growth; Adults: controls metabolism

Children growth usage @$10,000-$30,000/yrOne of first recomb proteins to be approved for human use

1985 Genentech Protropin; discontinued in late 90sProduced in E. coli and identical to native

Can re-engineer to augment of constrain mode of actionex, native HGH binds to GHR and prolactin RSide effects

Site-directed mutagenesis to change properties, ie structure/function


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Human growth hormone, optimization aslonger half-life

Relatively short half-life in plasma

Requires subcutaneous injection once a dayInconvenient and expensive

Method: fuse extracellular domain of HGHR, promotes dimerizationIn rats, promotes growth for 10 days [vs 1 day]Dimerization stabilizes and allows it to circulate 300x nativeMonomer is active and is slowly released from dimer


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Human growth hormone, optimizationlonger half-life

Relatively short half-life in plasmarequires subcutaneous injection once a dayinconvenient and expensive

Alternative method: fuse C-term with N-term of human serum albumin, Albutropinproduced in yeastgenetically modified to require minimal posttranslational modificationsserum half-life 19 days

Effective in rats and monkeys, 5 days after injection2009, Phase I clinical trials completed


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Pharmaceuticals: tumor necrosis factor alpha

TNF- is a potent antitumor agentNot widely used due to severe toxicity

Desire: deliver directly to site of action, less side effectsMethod: add Cys-Asn-Gly-Arg-Cys-Gly [targets a tumor cell surface protein]Mice: cytotoxic activities identical,

eg does not disrupt protein folding, trimerization, binding to receptor


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Tumor necrosis factor alpha: potential drug

TNF- with Cys-Asn-Gly-Arg_Cys-Gly [targets a tumor cell surface protein]Mice: cytotoxic activities identical,

eg does not disrupt protein folding, trimerization, binding to receptor

Modified version 12-15x more effective at inhibiting tumor growthHigher percentage of mice with lymphoma survived after treatment with modified versionMice that survived 30 days also survived a second and third challenge with lymphoma cells

2009, efficacy not yet demonstrated in humans


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Genetic disease: genomics and medicine

Cystic fibrosis, hereditary diseaseProgressive disability, early death [average life span 37 yrs]Difficulty breathing and lung infections, among symptoms

CF localized to cystic fibrosis transmembrane conductance regulatorRegulates components of sweat, digestive juices and mucous

One copy is enough for WT phenotype1 in 25 European descent carry a mutation, Ashkenazi Jews

http://en.wikipedia.org/wiki/Cystic_fibrosis


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Pharmaceuticals: Enzymes- DNaseI cloned

Cystic fibrosis patientsHighly susceptible to bacterial infections of lungs

Antibiotic treatments lead to resistanceAccumulation of mucous from bacteria-secreted alginate, DNA from bacteria and leukocytes

Genentech cloned DNaseI and expressed in CHO cellsDelivered as aerosol mist to lungs

1994 approved by FDA2000 sales of $100M


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Pharmaceuticals: Enzymes- DNaseI engineered

Actin binds very tightly to DNaseI

Inhibits enzymatic activity and limits therapeutic potential

Crystallographic data predicts residues to modifyAla-144 to Arg or Tyr-65 to Arg

Decreased actin binding up to 10,000x10-50x DNaseI activity

2009, clinical efficacy to be demonstrated


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Pharmaceuticals: Enzymes- alginase cloned

Alginate is a polysaccharide produced by seaweed, and soil and marine bacteriaCross-links to form gel with heavy viscosity

Pseudomonas aeruginosa excretes alginate into CF patient lungsProvides a biofilm that blocks antibiotics and provides anchorage

Alginate lysis gene from Flavobacterium species; cloned into E. coli


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Cloning alginate lyase

Selection/scoring methodsNot simply overexpress and observe activity

Flavobacterium sp.

Clone bank in E. coliScreen by plating onto medium plus alginate

Alginate lyaste requires cofactor Ca+++/- Ca++

Ca++ + alginate = cross-linked opaqueHydrolyzed alginate does not cross-linkAnalysis and characterization of clones and alginate lyase


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Alginate lyase[s]

ORF 69,000 DaPrecursor of three alginate lyases

-> 3,000 Da + 63,000 Da63,000 Da lyses both bacterial and seaweed alginates

63,000 Da -> 23,000 Da seaweed effective + 40,000 Da bacterial effectiveClone bacterial activity portion


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Alginate lyase[s]: Optimization of activity

Increase expression of 40,000 Da proteinPCR amplify and insertion behind strong promoter

B. subtilis plasmid, fused to a B. subtilis a-amylase leader peptide, directs secretion,and a strong penicillinase gene promoter

Expressed and assayed for halo phenotypeLiquifies alginates produced by P. aeruginosa isolated from lungs of CF patients2003, additional trials to determine if effective therapeutic agent


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State of the art, 2008-ish:Cystic fibrosis treatment follow-up

What is dornase alfa???


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State of the art, 2008-ish:Cystic fibrosis treatment follow-up

T t t f ti d t b li di Ph lk t i (PKU)


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Treatment of genetic and metabolic disease: Phenylketonuria (PKU)

Autosomal recessive genetic disorder in phenylalanine hydroxylase (PAH)Phe accumulation, decreases other large, neutral AAc in brain, needed for

protein and neurotransmitter synthesisBrain development; progressive mental retardation and seizuresIncidence ~1/15,000; But, varies: 1/4,500 Ireland and 1/100,000 Finland12q22-q24.1

Macaque genome: PAH gene sequence identical to a human PKU mutationwikipedia


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Phenylketonuria treatment[s]

Traditional treatment: diagnosis at birth or prenatal testControlled semi-synthetic diet with low levels of Phe

Possible treatment: metabolism of PhePAH multienzyme complex, requiring cofactor

Phe ammonia lyase (PAL)converts Phe [too] vsPAH

Stable and does not require cofactorTo test concept, yPAL cloned and overexpressed in E. coli

Preclinical studies (2003) with mice deficient in PALSee lower plasma levels of Phe when PAL injected or

administered as oral encapsulated enzyme

S h i d [ ]


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Smart therapies and treatment[s]:

Should we?

Traditional treatment: diagnosis at birth or prenatal testControlled semi-synthetic diet with low levels of Phe

Possible treatment: metabolism of PhePAH multienzyme complex, requiring cofactor

ELSI: Should we, just because we can?JGelsinger, 18yo (Sept 99)

Ornithine transcarbamylase deficiency, X-linked genetic diseaseCannot metabolize ammonia from protein catabolism

Usually fatal at birth, but this case was not inheritedSporadic and enough cells normal to allow life with diet and meds

First Gene Therapy-associated death in USThird childgene therapy in France developed cancer as a result


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Treatments for digestive

tract diseases

Ulcerative colitis, Crohn diseaseDiseases of intestinal tract~1/ 500-1,000

Ulcerative colitis- associated with excess type 2 T-helper cell cytokines, including Il-4 and -5Crohn disease- associated with excess type 1 T-helper cell cytokines, including TNF-, IFN-, IL-2

http://digestive.niddk.nih.gov/ddiseases/pubs/crohns/index.htm


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Smart therapies: Treatment with

live secreting bacteria

Ulcerative colitis- associated with excess type 2 T-helper cell cytokines, including IL-4 and -5Treatment: 1) antibodies against TNF-a, to lower levels of cytokines and 2) targeting IL-10

IL-10 modulates regulatory T-cells, that control inflammatory responses to intestinal AgDelivery is through injections directly or rectal enemasAlternative strategy: produce and deliver by intestinal bacteria

L. lactis to synthesize and secrete IL-10Mice fed water laced with dextran sulfate +/- recombinant L. lactis

Positive effect- Proof of PrincipleHowever, these mouse models not identicalto disease in humansConcern: recombinant bacteria released into environment


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Treatment with live secreting

bacteria

Concern: recombinant bacteria released into environmentMethod: synthetic human interleukin-10 gene replaces L. lactis thymidylate synthetase gene [thyA]

Essential for bacterial growthEntire construct recombined into chromosomeProduces human interleukin-10 and grows well if thymidine or thymine is provided

Tested in pig [digestive tract ~ human]Extremely unlikely L. lactis will pick up thyA from environment

2009, clinical trials with 10 Crohns patientsBacteria isolated in feces are thyA-


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Smart therapies: Treatment of obesity

30% North Americans and 20% Europeans are overweight10s of $B spent on weight reduction schemes

Leptin: 16 kDa protein hormone, regulates Energy uptake/expeditureAppetite and metabolism; satiety- appetite control

1950 Jackson Lab: mutant obese mice, massively obese and excessively voraciousSeveral strains homozygous for single-gene mutationsTwo classes: ob/ob, leptin mutations and db/db, leptin receptor mutations

ob/ob mice + injected leptin => lose excess fat and return to normal body weight1995 leptin gene, EDGreenJMFriedman Genome Research 5:5-12

Acts on hypothalamus of brain http://en.wikipedia.org/wiki/File:Fatmouse.jpghttp://en.wikipedia.org/wiki/Leptin


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Smart therapies: Treatment of obesity

30% North Americans and 20% Europeans are overweight10s of $B spent on weight reduction schemesTreatment with recombinant leptin can reduce food intake and correct

metabolic perturbations in ob/ob miceCan help human congenital leptin deficiency

Subcutaneously, not particular effective unless serum conc 20-30x higher than normalBlood-brain barrier

Method: intranasal delivery of leptinLeptin is synthesized as precursor molecule with 21-amino acid signal peptideE. coli product insoluble -> solubilize

Time-consuming, inefficient and expensiveNisin promoter and deliver using L. lactis

No inclusion body and secretedIntranasal administration significantly reduced food intake and body weight in obese mice


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Therapeutic agents from Transgenic animals, Molecular biology, Genomics


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http://www.usatoday.com/news/health/2008-10-26-PTSD-main_N.htm

Post-traumatic stress (PTS) disorderTreating a mental disorder?Once poorly understood and

Little-known mental health problemBetter known as war vets seek treatment

Rate of 1 per 7 back from deploymentMany myths

1- psychologicalbiologically-based

2- military combat is top causecar accidents is top cause

Long-term effects if not treatedCan develop other problems, eg addictionsTreatment can include therapy and medication


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http://www.usatoday.com/news/health/2008-10-26-PTSD-main_N.htm

Post-traumatic stress (PTS) disorderUSAToday, Oct 27, 2008Many myths

1- psychologicalbiologically-based

2- military combat is top causecar accidents is top cause

Long-term effects if not treatedWith PTSD, an average of 12 years before seeking treatment after accident

2.5M in US injured per year in car accidentsCan develop other problems, eg addictionsTreatment can include therapy and medicationSSRI-class antidepressants

(selective serotonin reuptake inhibitor)


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The Scientist, Oct 22, 2008

Proteins and memory, and mice

XCaoJZTsien, et al. Neuron Oct08Memory is separated into four stages

Acquisition, consolidation, storage and retrievalSpecific proteins play roles in these phases

One memory molecule is Calcium/calmodulin-dependent protein kinase II (CaMKII)

Plays key role in brain cell communication; linked to learning and memoryCreated transgenic mice with inducible promoter hooked to CaMKIIExamined transgenic mice for retrieval ofshort-term and long-term fear memoriesand novel object recognition memory

(fear= mild electric shock and cat odor causes mice to freeze in the contextof a specific environment)


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Memory, molecular biology and cinema

NPR 4/13/10 Hippocampus: memory and lingering feelingsEmotions outlast the memories that drive them; JFeinsteinDTranel PNAS pPub Apr10Alzheimers patients, visits/moods, post-visit: no memory but depressed or elated feeling remains

i d


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Protein and memory,and mice

XCaoJZTsien, et al. Neuron Oct08Transgenic with CaMKII activity that can be inducibly and reversibly switched offand on within minutes by injection of a genetically sensitized inhibitor, NM-PP1 ->

Transient overexpression of CaMKII at time of recall can lead to rapid erasureof a memory that is being retrieved, leaving other memories intact

Also memories formed in the past hourEffect persists for a month after

Applications include PTS treatment andUnderstanding memory circuits in the brainEarlier work on memory and protein

P i d d i


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Protein and memory, and mice

YPTangJZTsien, et al. Nature Sept991999 Doogie mouse- smart transgenic mouse

with enhanced learning and memory abilitiesNR2B gene, encoding NMDA receptor

Two chemicals needed to trigger this nerve cell receptorMice with extra NR2B genes have heightened NMDA receptor activityAdding a single gene to fertilized eggsAssay by environment containing two objects, then removed one

Observe how long spent at new object versus old: test memoryAssay with environment and electric shock: test learningAssay with pool and hidden ramp: test spatial intelligenceApplication: Gene therapy for dementia

Earlier work, limiting expression of NR2B could impair ability to learn and rememberDeletion of the gene in certain brain regions resulted in mice that were considerably less intelligent

http://www.sciam.com/article.cfm?id=making-smart-micewiki

P i d d i


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Protein and memory, and mice

http://www.princeton.edu/pr/pictures/other/smartmouse/index.html

Ch i d i


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Chemistry, sex, and mice

http://www.bbc.co.uk/news/health-12825688http://www.nature.com/jp/journal/v25/n9/fig_tab/7211352f1.htmlhttp://www.nature.com/nature/journal/vaop/ncurrent/full/nature09822.html

Serotonin controls sexual preference in mice [Liu, Jiang, Kim, et al., March 2011 Nature]1) Males bred not receptive to serotonin lose preference for females

Mount and emit mating call

2) Different mutation, lacking tryptophan hydroxylase 2 gene (precursor to serotonin)

Could restore preference by injecting serotonin into brainMouse sexual preference linked to smellHumans: limited evidence for altered responses to selective serotonin uptake inhibitors (SSRIs)Psychoactive drugs that increase or decrease serotonin function =>

sexual arousal, impulsivity and aggression

Th B i


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The Brain

http://en.wikipedia.org/wiki/Phineas_Gage

Phineas P. Gage 1823-1848-1860Left frontal lobe destroyedEffects on personality and behaviorFriends said no longer GageFirst case to suggest specific brain damage and personality and behavior

Th B i i b h i t h i l l ti


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The Brain: mapping behavior to physical location

http://www.headinjury.com/brainmap.htm

Damage to hippocampus interferes with new memory storageAlso, use of language, recognize familiar faces, count and read



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Frontal lobes



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Temporal lobes



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Parietal lobes



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Occipital lobes

G i G ti i ti


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Genomics: Genetic vs epigenetic-unusual symptoms/syndromes (new understandings)

10/28/06 Scott Adams. Rare example of recovery- largely but not totallySpasmodic Dysphonia, mysterious disease in which parts of the brain

controlling speech shut down or go haywire30,000 Americans, typically in 40s and 50sPhenotype: Typically unable to converse in normal voice, but under

different circumstances, immediately after sneezing or laughing, can speak in exaggerated falsetto or baritone, or while reciting poetry

Off-label drug use: Botox

Genomics: Genetic vs epigenetic


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Genomics: Genetic vs epigenetic-

unusual symptoms/syndromes (new understandings)

Diane Rehm, Spasmodic Dysphonia, diagnosed in 1998Cause unknownMay run in families (inherited)

Chrom 9 region/gene affecting spasms of the vocal cordsAlso spasms in eyes, arms, legs and mouthAl h l i l d i di d

08 engr prot therapy032911

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