Biotechnology

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5 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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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



Protein engineering

2003

1000s of enzymes studied and characterized biochemically

20s account for >90% 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



2003 enzymes, “industrialized”



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?



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]



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



Refolding insoluble
overexpressed proteins



Changing Asn to other AAc

At high temperatures, Asn and Gln may undergo deamidation

Converting to Asp and Glu

Localized changes in structure-> function?

S. cerevisiae triosephosphate isomerase, homodimer

Asn->Asp, lose half-life, lose activity as well

Correlation between temperature stability and protease-resistance



Reducing number of free sulfhydryl residues

Expressed recombinant protein may be

less active than native or expected

Protein engineered to increase activity

ex., human
β
-interferon (IFN-
β
)

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
β
, but data on
α

Use to deduce which
β
Cys to mutate (Cys17)

Mutant has similar SA as native and more stable in

Longer-term storage than native



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 --> Tyr-A + PP
i
(2)
Tyr-A + tRNA
Tyr
--> Tyr-tRNA
Tyr
+ 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



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,
α
helix portion



Metal cofactor requirement

Modification of proteins: changing requirements

holoenzyme <--> apoenzyme

Metal cofactors

ex., subtilisins, serine proteases

Excreted by gram positive bacteria

->Biodegradable cleaning agents, laundry detergent

Requires Ca
++
as cofactor

Ka =10
7
M

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



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 ->

10x more stable than native form sans Ca
++
/50% more stable in Ca
++



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



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



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

>2,500 REs known, only 200 different recognition sites

4-6 bp cutters not as useful as >8 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]



Modifying antibodies

Immunoglobulins

Light chain plus heavy chain; di-sulfide bonds

Hypervariable portion determines specificities

wikipedia



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
>
1 AAc changes specificity

Random mutagenesis with degenerate oligo primers gives range of different mutations



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?



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



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??]]



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



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




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]
Laundry, detergent and mushrooms

http://www.fotosearch.com

http://www.education.umd.edu/EDMS/mislevy/Drawings/washing.jpe

http://www.wildaboutbritain.co.uk/gallery/g




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 -> 174x thermal stability and 100x oxidative stability

Cherry…Pedersen. 99. Nat Biotech “Directed evolution of a fungal peroxidase”
Mushroom peroxidase


Molecular analysis of hybrid peroxidase






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



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



The Industrial Age

Jose Maria Sert “American Progress, the Triumph of Man’s

Accomplishments Through Physical and Mental Labor”

GE Bldg, Rockefeller Center. 1937

1930’s deco: “Art and Power”

[Man can change Nature]



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,

-> combination of enzymes that reduces cellulosic biomass into fermentable sugars

Synthetic Genomics, Rockville-based, is searching for naturally occurring cellulases

WaPost



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) -> 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



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/blaspirin.htm



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



Natural Products: Tamoxifin

Tamoxifen, orally active selective estrogen receptor modulator (SERM)


Treatment of breast cancer (currently the world’s 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



Natural Products: plant sterols

β
-sitosterol, plant sterol

Induces apoptosis and activates key caspases in MDA-MB-231 human breast cancer cells

ABAwad, RRoy, CSFink. Oncology Reports 10:497 (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



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



Natural Products: Bioprospecting

WLSmith and WCWheeler. 2006.

NYTimes



Recombinant proteins for human use

~2003

Approved in US or EU


Recombinant interferon:
isolation of cDNA

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
6,000 clones



Hybrid products: INF

IFN cDNA isolated early 80s

Now, three groups of IFN genes identified:
α
,
β
,
γ

IFN
α
family of 13 genes; IFN
β
family of 2 genes; IFN
γ
of 1 genes

Subtypes have different specificities

IFN
α
1 and
α
2 have similar antiviral activities when assessed with virus-challenged bovine cell line

IFN
α
2 is 7x more effective than
α
1 when human cells treated with virus

IFN
α
2 is 30x less effective than
α
1 when mouse cells treated with virus

IFN
α
1 and
α
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



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



Recombinant modification: hTNF-
α

Tumor necrosis factor alpha (TNF-
α
)

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



Recombinant modification: hTNF-
α

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)?



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



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-
α
, IFN-
α
, IL-2
http://digestive.niddk.nih.gov/ddiseases/pubs/crohns/index.htm



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



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 -> 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



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



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)



Clearing the lungs 2 with alginate lyase

http://www.lsbu.ac.uk/water/hyalg.html

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



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



Alginate lyase[s]

ORF 69,000 Da

Precursor of three alginate lyases

-> 3,000 + 63,000

63,000 lyses both bacterial and seaweed alginates

63,000 -> 23,000 seaweed effective+ 40,000 bacterial effective

Clone bacterial activity portion



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



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



Phenylketonuria treatment[s]

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