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