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Title: Biotechnology


1

Lecture 7 Human applications of the products of
molecular biotechnology
  • (Protein engineering (Chapt 8))
  • Therapeutic agents (Chapt 10)
  • Monoclonal antibodies (Chapt 10)
  • Gene therapy (Chapt 10)
  • Molecular diagnostics

2

Protein engineering
  • 2003
  • 1000s of enzymes studied and characterized
    biochemically
  • 20s account for gt90 enzymes used industrially
  • Native protein do not meet the needs of highly
    specialized industrial applications
  • Most denatured by conditions required
  • High temperature, organic solvents
  • Thermotolerant organisms may not have appropriate
    counterpart enzymes

3

2003 enzymes, industrialized
4

One immediate goal, thermal stability
  • Thermostability may result in organic solvent and
  • non-physiological conditions stabiity (pH)
  • Addition of di-sulfide bridges
  • But, does it affect function?
  • That is, removing the original AAc? Adding Cys?
    Adding S-S?

5

Example T4 lysozyme
  • T4 lysozyme
  • Originally, no S-S bonds
  • 1) Pseudo-WT demonstrates existing Cys do not
    have functional role
  • 2) Site-directed mutagenesis to add S-S
  • 3) Add multiple S-S bonds
  • Results Some good, some better Some cases,
    loss of activity
  • due to corruption of original structure

6

Example 2 ribonuclease
  • Bull semen RNase can act as anti-tumorigenic
    agent
  • In vitro and in vivo, dimeric form is
    internalized into tumor cells by
  • non-receptor-mediated endocytosis
  • In cytosol, degrades rRNA, blocking translation
    and cell death occurs
  • Human anti-bull semen Rnase Ab limits use in
    human trials
  • Human is 70 identical to bull semen RNase
  • Cloned, engineered human RNase in E. coli is
    insoluble
  • Renatured human version has less anti-tumorigenic
    activity
  • TBD

7

Refolding insoluble overexpressed proteins
8

Changing Asn to other AAc
  • At high temperatures, Asn and Gln may undergo
    deamidation
  • Converting to Asp and Glu
  • Localized changes in structure-gt function?
  • S. cerevisiae triosephosphate isomerase,
    homodimer
  • Asn-gtAsp, lose half-life, lose activity as well
  • Correlation between temperature stability and
    protease-resistance

9

Reducing number of free sulfhydryl residues
  • Expressed recombinant protein may be
  • less active than native or expected
  • Protein engineered to increase activity
  • ex., human ?-interferon (IFN-?)
  • Expressed in E. coli
  • 10 antiviral activity of native glycosylated
    form
  • Also, most expressed as inactive dimers and
    multimers
  • Note three Cys that were not S-S in native
  • Use Ser to substitute for Cys -OH for -SH
  • No data on ?, but data on ?
  • Use to deduce which ? Cys to mutate (Cys17)
  • Mutant has similar SA as native and more stable
    in
  • Longer-term storage than native

10

Increasing enzymatic activity
  • Modify catalytic function by site-directed
    mutagenesis
  • One method is to modulate substrate-binding
    specificity
  • B. stearothermophilus tyrosyl-tRNA synthase
  1. Tyr ATP --gt Tyr-A PPi
  2. Tyr-A tRNATyr --gt Tyr-tRNATyr AMP
  • Both reactions occur while substrates are bound
    to enzyme
  • Have 3-D structure, mapped active site
    biochemical data
  • Thr51 replaced by Ala or Pro
  • Native enzyme Thr forms a weak H-bond with Tyr
    ring
  • Removal may increase affinity for ATP

11

Increasing enzymatic activity
  • Results
  • Thr to Ala, binds better, 2x, with similar
    activity
  • Thr to Pro, binds 100x better, with higher
    activity
  • Unexpected, Pro should have altered structure
    dramatically, ? helix portion

12

Metal cofactor requirement
  • Modification of proteins changing requirements
  • holoenzyme lt--gt apoenzyme
  • Metal cofactors
  • ex., subtilisins, serine proteases
  • Excreted by gram positive bacteria
  • -gtBiodegradable cleaning agents, laundry
    detergent
  • Requires Ca as cofactor
  • Ka 107M
  • Stabilizes protein structure
  • Industrial setting large number of
    metal-chelating
  • conditions
  • Two-step enhancement
  • 1) abolish Ca binding
  • 2) increase stability of protein
  • B. amyloliquefaciens subtilisin BPN
  • 3-D structure biochem characterized
  • Delete 75-83 abolishes Ca binding
  • retains structure

13

Metal cofactor requirement
  • Step 2 restoring functionality
  • Ten AAc interacted with deleted Ca-binding loop
  • Which contributes to native 3-D structure
  • Four domains identified, and modified
  • Assay grow mutants, heat to 65C, test
    subtilisin activity
  • lethal in E. coli, use B. subtilis
  • Results see 7/10 positives combine into one -gt
  • 10x more stable than native form sans Ca/50
    more stable in Ca

14

Decreasing protein sensitivity
  • Streptococcus streptokinase, 47 kDa protein that
    dissolves blood clots
  • Complexes 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

15

Decreasing protein sensitivity
  • Streptococcus streptokinase, plasmin sensitivity
    domain
  • Attacks at Lys59 and Lys382, near each end of
    protein
  • Resultant 328 AAc peptide has 16 activity
  • Mutate Lys to Gln
  • Gln has similar size/shape to Lys also no charge
  • Single mutations similar to double to native in
    binding and activating plasminogen
  • In plasmin presence, half-lives increased with
    double as 21x more resistant to cleavage
  • TBD longer life wanted

16

Modifying protein specificity
  • Previous protein engineering focused on modifying
    and enhancing existing properties
  • Conceivable to redesign enzyme with new unique
    catalytic activity
  • ex., new site-specific endonucleases designed
    from FokI, Flavobacterium okeanokoites
  • gt2,500 REs known, only 200 different recognition
    sites
  • 4-6 bp cutters not as useful as gt8 bp cutters
    engineer this rather than screen for new RE
  • Zn-finger proteins that binds to DNA major groove
  • Mouse protein Zif268 has three separate Zn-finger
    domains, binding to DNA independently
  • Construct His tag for purification three
    Zn-finger domains nuclease domain
  • Two versions, one cuts at target site other cuts
    at expected site plus two related sites
  • Zn-finger domains recognize triplet codes but
    interact with two of the three bases

17

Modifying antibodies
  • Immunoglobulins
  • Light chain plus heavy chain di-sulfide bonds
  • Hypervariable portion determines specificities
  • wikipedia

18

Modifying antibodies
  • Hypervariable portion determines specificities
  • Can truncate Ab to Fab fragment, with binding
    activity
  • CDR hypervariable complementarity-determining
    region
  • FR framework region
  • Six total CDRs, one set from H and one from L
    chains
  • Altering gt1 AAc changes specificity
  • Random mutagenesis with degenerate oligo primers
    gives range of different mutations

19

Modifying antibodies, error-prone PCR
  • Protocol
  • One CDR of heavy chain modified by error-prone
    PCR
  • Second PCR- other two CDRs modified by
    error-prone PCR
  • Third, PCR all three modified CDRs into one heavy
    chain
  • ex., mAb Fab for 11-deoxycortisol altered to bind
    only to cortisol
  • TBD to any Ag determinant?

20

Modifying two properties Increasing enzyme
stability and specificity
  • Tissue plasminogen activator (tPA)
  • Multidomain serine protease
  • Medically useful for dissolving blood clots
  • Rapidly cleared from circulation, so needs to be
    transfused
  • Needs to be used as high concentrations at the
    start
  • Side effect nonspecific internal bleeding
  • Need 1) long-lived tPA with 2) increased
    specificity to fibrin in blood clot, and 3) no
    internal bleeding
  • Solution directed mutagenesis

21

Modifying two properties Increasing enzyme
stability and specificity
  • Site-directed mutagenesis
  • 1) Thr103 to Asn, half-life extended in rabbit
    plasma, 10x longer than native
  • 2) 296-299 to Ala string, more specific for
    fibrin
  • 3) Asn117 to Gln, retains level of fibrinolytic
    activity of original
  • Combination of all three, expressed all three
    phenotypes
  • TBD is modified form tPA suitable replacement
    for native?
  • side effect??

22

Altering multiple properties simultaneously
  • Properties useful in an industrial process often
    do not exist in Nature
  • ex., highly active at 23C and stable at 70C
  • Modifying one property may disrupt other
    properties, some critical
  • Molecular breeding of new proteins, using
    several similar genes
  • using DNA shuffling protocol
  • Does not require prior knowledge of
    structure/function of target protein
  • ex., subtilisin
  • Use 26 different subtilisin genes
  • Shuffle DNA, construct library of 654 clones, and
    Tf B. subtilis to hardcopy
  • Assay in microtiter plates

23

Altering multiple properties rapid
high-throughput screening
  • ex., subtilisin
  • Use 26 different subtilisin genes
  • Shuffle DNA, construct library of 654 clones, and
    Tf B. subtilis
  • Assay in microtiter plates originals plus
    clones
  • Activity at 23C thermostability solvent
    stability pH dependence
  • Of 654 clones, 77 versions performed as well as
    or better than parents at 23C
  • Sequencing showed chimeras one has 8 crossovers
    with 15 AAc substitutions

24

Laundry, detergent and mushrooms
  • First to combine two site-directed mutagenesis
    techniques with gene shuffling and sorting
    procedures
  • Directed evolution
  • JCherry 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
  • http//www.fotosearch.com
  • http//www.education.umd.edu/EDMS/mislevy/Drawings
    /washing.jpe
  • http//www.wildaboutbritain.co.uk/gallery/g

25

Mushroom peroxidase
  • ex., Coprinus cinereus heme peroxidase (ink cap
    mushroom) 343 AAc, heme prosthetic group
  • Multiple rounds of directed evolution to generate
    mutant for dye-transfer inhibitor in laundry
    detergent
  • Native form or WT is rapidly inactivated under
    laundry conditions at pH 10.5,
  • 50C and high peroxide concentrations (5-10mM)
  • Combined mutants from site-directed and random
    mutagenesis led to mutant with
  • 110x thermal stability, 2.8x oxidative stability
  • Additional in vivo shuffling of pt mutations -gt
    174x thermal stability and 100x oxidative
    stability
  • CherryPedersen. 99. Nat Biotech Directed
    evolution of a fungal peroxidase

26
Molecular analysis of hybrid peroxidase
27

28

Therapeutic agents
  • Prior to recombinant DNA technology, most human
    protein pharmaceuticals were available
  • in limited quantities
  • Costly to produce, modes of action not well
    characterized
  • Evolution of therapeutic agents
  • Natural products
  • Accidental discovery/use of mixtures to
  • isolation/use to
  • synthesis by Nature to
  • Organic Chemistry (Age of Industrialization) to
  • proteins (and antibodies) to
  • recombinant DNA technology (Molecular
    cloning/Protein engineering) to
  • Bioprospecting

29

Therapeutic agents
  • Horse/cow sera- antibodies influenza vaccine
  • Blood donors- blood, bood components (clotting
    agents/hemophilia)
  • Cadavers- human growth hormone from pituitary
    glands
  • 1985. Genentech- FDA approval to sell first
    biotech industry product,
  • recombinant human growth hormone vs
    cadaver-derived product
  • Animal sources- porcine insulin prior to 1982
    then recombinant human insulin

30

The Industrial Age
  • Jose Maria Sert American Progress, the Triumph
    of Mans
  • Accomplishments Through Physical and Mental
    Labor
  • GE Bldg, Rockefeller Center. 1937
  • 1930s deco Art and Power
  • Man can change Nature

31

Bioprospecting Microbes with desired product or
function
  • 1997. Soil sampling at Quabbin Reservoir, Boston
    (TWarnick, UMAmherst)
  • 2007. Isolate that degrades cellulose, producing
    ethanol (SBLeschine, UMAmherst)
  • Also as Chief Scientist at SunEthanol, start-up
    biotech Q microbe does both in one organism
  • (SunEthanol/DOE sequencing) (working with 1-2 L
    in lab to large-scale)
  • Naturally occurring vs in vitro synthesized
    components
  • (last lectures, recombinant DNA technology and
    Protein engineering)
  • Genencor, division of Danisco, 2007 announced dev
    of new product Accellerase 1000,
  • -gt combination of enzymes that reduces
    cellulosic biomass into fermentable sugars
  • Synthetic Genomics, Rockville-based, is searching
    for naturally occurring cellulases
  • WaPost

32

Aspirin Natural Products
  • Salicylic acid is a phytohormone and a phenol,
    ubiquitous in plants
  • Plant growth and development, photosynthesis,
    transpiration, ion uptake and transport
  • Leaf anatomy and development, chloroplast
    structure
  • Endogenous signal mediating plant defense against
    pathogens
  • Willow (Salix) -gt Spiraea
  • Hippocrates, 460-377 B.C., was left historical
    records of pain relief treatments,
  • Use of powder made from bark and leaves of the
    willow to treat headaches, pains and fevers
  • 1829, salicin in willow trees
  • Bayer
  • wikpedia

33

Aspirin Age of Industrialization (Organic
Chemistry)
  • Acetylsalicylic acid, derivative of
  • Salicylic acid- mild nonnarcotic analgesic
  • Inhibits prostaglandins, nec for blood clotting
    and sensitize nerve endings to pain
  • Isolated and purified, characterized, synthesized
    by several scientists
  • 1899, Felix Hoffman at Bayer rediscovered
    buffering formula of Gerhardt (1953)
  • 1915, available in tablet form

http//inventors.about.com/library/inventors/blasp
irin.htm
34

Penicillin Natural Products
  • Ancient Greece, India- molds and plants to
    treat infection China- moldy bean curd on cuts
  • 1929. AFleming, Penicillium mold must have an
    antibacterial substance
  • Isolated and named active substance, penicillin,
    from halo of inhibition of bacterial growth
  • around a contaminant blue-green mould on a
    Staphylococcus plate culture.
  • Unsuccessful attempts to recruit chemist to
    synthesize for mass production
  • HWFlorey et al (1938)/Moyer, Coghill, Raper
    (1941-3)/JKane, Pfizer scientists (1941-4)
  • Large quantities of pharmaceutical-grade
    penicillin
  • wikipedia

35

Natural Products Tamoxifin
  • Tamoxifen, orally active selective estrogen
    receptor modulator (SERM)
  • Treatment of breast cancer (currently the
    worlds largest selling drug for this)
  • For early and advanced ER (estrogen receptor
    positive) breast cancer
  • Screened as a morning-after contraceptive
    drug/ALWalpole/ICI Pharmaceuticals
  • 1962 ICI/DRichardson synthesized ICI-46,474
  • 1971 clinical study at Christie Hospital
    advanced breast cancer
  • wikipedia

36

Natural Products plant sterols
  • ?-sitosterol, plant sterol
  • Induces apoptosis and activates key caspases in
    MDA-MB-231 human breast cancer cells
  • ABAwad, RRoy, CSFink. Oncology Reports 10497
    (2003)
  • Caspases play roles in apoptosis Caspase 3
    fragments DNA. Caspase 8, initiator for an
  • extrinsic pathway, and Caspase 9, initiator for
    intrinsic pathway, both activate Caspase 3

37

Natural Products Bioprospecting
  • WLSmith and WCWheeler. 2006.
  • 1,200 species of venomous fish
  • JHeredity, Venom evolution widespread in
    fishes
  • Stung by spines of dead fuzzy dwarf lionfish
    passed out as reached into trashcan
  • Fish venom blood clotting, nerve and muscle
    activity, blood pressure and heartbeat
  • Wikipedia
  • J Heredity

38

Natural Products Bioprospecting
  • WLSmith and WCWheeler. 2006.
  • NYTimes

39

Recombinant proteins for human use
  • 2003
  • Approved in US or EU

40
Recombinant interferon isolation of cDNA
6,000 clones
  • Strategies for isolating either the genes or
    cDNAs for human proteins
  • 1) Isolate target protein and determine partial
    AAc sequence
  • Synthesize oligo as probe to screen cDNA library
  • 2) Generate Ab against purified proteins
  • Screen gene library
  • Interferon strategy above, pre-human genome
    sequence

41

Hybrid products INF
  • IFN cDNA isolated early 80s
  • Now, three groups of IFN genes identified ?, ?,
    ?
  • IFN? family of 13 genes IFN? family of 2 genes
    IFN? of 1 genes
  • Subtypes have different specificities
  • IFN ?1 and ?2 have similar antiviral activities
    when assessed with virus-challenged bovine cell
    line
  • IFN ?2 is 7x more effective than ?1 when human
    cells treated with virus
  • IFN ?2 is 30x less effective than ?1 when mouse
    cells treated with virus
  • IFN ?1 and ?2 have common RE sites
  • Hybrid INFs demonstrate potential therapeutics by
    combining functional domains
  • Some (2003)- successful clinical trials, approved
    for use as human therapeutic agents

42

Site-specific directed mutagenesis hGH
  • hGH 191 AAc, 22,1 kDa
  • One of first therapeutic proteins approved for
    human use
  • Recombinant form produced in E. coli, identical
    to native pituitary-derived hGH
  • Native binds to growth hormone receptor and
    prolactin receptor
  • Side effects
  • Prolactin receptor binding function of Zn
    binding
  • Domain His-18, His-21, Glu-174
  • 2003, testing mutants

43

Recombinant modification hTNF-?
  • Tumor necrosis factor alpha (TNF-?)
  • Potent antitumor agent
  • Not widely used due to severe toxicity
  • If can be delivered directly to site of action,
    then lower doses and less side effects
  • Develop version with tumor specificity
  • Fusion Cys-Asn-Gly-Arg-Cys-Gly at N-terminus
  • In mice, cytotoxic activities identical
  • ie, does not affect folding, trimerization,
    receptor binding
  • Modified version 12-15x more effective at
    inhibiting tumor growth

44

Recombinant modification hTNF-?
  • Fusion Cys-Asn-Gly-Arg-Cys-Gly at N-terminus
  • In mice, cytotoxic activities identical
  • ie, does not affect folding, trimerization,
    receptor binding
  • Modified version 12-15x more effective at
    inhibiting tumor growth
  • Greater percentage of mice with lymphoma survived
    after treatment
  • Also, 30-day survivors able to survive second and
    third challenge with mouse lymphoma cells
  • Efficacy in humans (2003)?

45

Optimizing gene expression
  • Multistep process
  • Design a protein, construct a recombinant
    molecule, express and characterize
  • Need to optimize expression
  • First, either prokaryote or eukaryote host
  • Comparative analysis of host and expression
  • ex., interleukin-3 expression
  • Best in B. licheniformis
  • Balance with glycosylation in eukaryotic hosts
  • But, glycosylation is not essential for
    interleukin-3 activity

46

Treatments for digestive tract diseases
  • Ulcerative colitis, Crohn disease
  • Diseases of intestinal tract
  • 1/ 500-1,000
  • Ulcerative colitis- associated with excess type 2
    T-helper cell cytokines, including Il-4 and -5
  • Crohn disease- associated with excess type 1
    T-helper cell cytokines, including TNF-?, IFN-?,
    IL-2

http//digestive.niddk.nih.gov/ddiseases/pubs/croh
ns/index.htm
47

Treatment with secreting bacteria
  • Ulcerative colitis- associated with excess type 2
    T-helper cell cytokines, including IL-4 and -5
  • Treatment 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 Ag
  • Delivery is through injections directly or rectal
    enemas
  • Alternative strategy produce and deliver by
    intestinal bacteria
  • L. lactis to synthesize and secrete IL-10
  • Mice fed water laced with dextran sulfate /-
    recombinant L. lactis
  • Positive effect- Proof of Principle
  • However, these mouse models not identical to
    disease in humans

48

Cystic fibrosis
  • Genetic disease affecting lungs and digestive
    system
  • Average life span 37 years, extended and
    extending
  • In US, 1/3,900 1/22 are carriers
  • Most common in Europeans and Ashkenazi Jews
  • Cystic fibrosis transmembrane conductance
    regulator (CFTR)
  • Chloride ion channel, sweat, digestive juices and
    mucus
  • thick, sticky mucus to build up in the lungs and
    digestive tract
  • 7q31.2 -gt 180,000 bp gene, 1,480 AAc
  • Most common mutation DF508 1,400 other mutations
  • DF508 missense, not folded correctly
  • Lungs susceptible to bacterial infection
  • Antibiotics treatment results in resistance and
  • combination with DNA from bacteria and
    leukocytes causes pulmonary problems (mucus)
  • wikipedia

49

Treatment
  • Genentech hDNase I in CHO cells
  • Not a cure, but alleviates symptoms
  • Purified protein delivered via aerosol mist to
    lungs of CF-
  • Approved by FDA in 1994

50

Optimizing treatment
  • In response to bacteria in lungs,
  • leukocytes cluster and lyse bacteria (and
    leukocytes)
  • Lysed leukocytes release actin
  • Monomeric actin binds DNase I very tightly and
    inhibits
  • Limits effectiveness
  • X-ray structure data suggested Ala-144 required
    for binding
  • or Tyr-65
  • Changing either to Arg decreases actin binding by
    10,000x
  • Clinical efficacy of mutants to be determined
    (2003)

51

Clearing the lungs 2 with alginate lyase
  • Alginate produced by seaweeds, soil and marine
    bacteria
  • P. aeruginosa excretion in lungs contributes to
    viscosity of mucus
  • In addition to DNase I treatment, alginate lysate
    can be used as therapeutic agent
  • Flavobacterim sp., gram-negative soil bacterium
  • http//www.lsbu.ac.uk/water/hyalg.html

52

Cloning alginate lyase
  • Flavobacterium sp.
  • Clone bank in E. coli
  • Screen by plating onto medium plus alginate
  • /- Ca
  • Ca alginate cross-linked opaque
  • Hydrolyzed alginate does not cross-link
  • Analysis and characterization of clones and
    alginate lyase

53

Alginate lyases
  • ORF 69,000 Da
  • Precursor of three alginate lyases
  • -gt 3,000 63,000
  • 63,000 lyses both bacterial and seaweed alginates
  • 63,000 -gt 23,000 seaweed effective 40,000
    bacterial effective
  • Clone bacterial activity portion

54

Optimization of activity
  • Increase expression of 40,000 protein
  • PCR amplify and insertion behind strong promoter
  • B. subtilis plasmid, fused to a B. subtilis
    a-amylase leader peptide, directs secretion and
  • penicillinase gene promoter
  • Expressed and assayed for halo phenotype
  • Liquifies alginates produced by P. aeruginosa
    isolated from lungs of CF patients
  • 2003, additional trials to determine if effective
    therapeutic agent

55

Phenylketonuria (PKU)
  • Autosomal recessive genetic disorder in
    phenylalaniine hydroxylase
  • Phe accumulation, decreases other large, neutral
    AAc in brain, needed for
  • protein and neurotransmitter synthesis
  • Brain development progressive mental retardation
    and seizures
  • Incidence 1/15,000 varies 1/4,500 Ireland and
    1/100,000 Finland
  • 12q22-q24.1
  • Macaque genome PAH gene sequence identical to a
    human PKU mutation
  • wikipedia

56

Phenylketonuria treatments
  • Traditional treatment diagnosis at birth or
    prenatal
  • Controlled semi-synthetic diet with low levels of
    Phe
  • Possible treatment metabolism of Phe
  • PAH multienzyme complex, requiring cofactor
  • Phe ammonia lyase (PAL) converts Phe as well
  • Stable and does not require cofactor
  • To test concept, yPAL cloned and overexpressed in
    E. coli
  • Preclinical studies (2003) with mice deficient in
    PAL
  • See lower plasma levels of Phe when PAL injected
    or
  • administered as oral encapsulated enzyme
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