Lecture 7 - Genome Tools

Mammals as bioreactor 

•Use of animals as ‘factories’ for production of bio-products of human needs 

•Proteins 

•Organs 

•Tissues

GFP and whole animal expression

•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/ 

Tumor studies in vivo, in situ; 

specific patterns, spatial and temporal 

•Application of GFP as marker

“Green Revolution” 

•Nobel 08, Biology 

•Montagnier, Barre-Sinoussi (HIV); Hausen (HPV) 

•Nobel 08, Chemistry 

•Roger Tsien, Martin Chalfie, Osamu Shimomura 

•“Green fluorescent protein (GFP)” 

•“…impact of GFP on science to the invention 

of the microscope… the protein has been 

‘a guiding star for biochemists, biologists, 

medical scientists and other researchers’.” 

•http://www.chinadaily.com.cn/china/2008-10/09/content_7088785.htm 

•http://www.npr.org/templates/story/story.php?storyId=95504782


•http://www.npr.org/templates/story/story.php?storyId=95504782 

“Color Revolution” 


•When exposed to uv light, protein glows green 

•Used as a tracer to expose movement 

•Mark particular cells in a tissue 

•Shows when and where particular genes turn on and off 

•“The Brainbow:” ‘spectacular experiment tags different nerve 

cells in the brain of a mouse’ 

•“Nature Nov 1, 2007 “Over The Brainbow” 

•~100 yrs ago Ramon Y Cajal stain Golgi on nerve cells- door to modern neurobiology 

•Could see previously invisible axons and dendrites as they moved in the tissue 

•‘Now, a method to visualize many distinct cells within a brain circuit’ 

•JLivet…JWLichtman. “Transgenic strategies…in the nervous system”


•http://www.npr.org/templates/story/story.php?storyId=95504782 

“Green Revolution” 


•When exposed to uv light, protein glows green 

•Used as a tracer to expose movement 

•Mark particular cells in a tissue 

•Shows when and where particular genes turn on and off 

•“The Brainbow:” ‘spectacular experiment tags different nerve 

cells in the brain of a mouse’ 

•Shimomura and a colleague (Woods Hole, MA and BU Medical School) 

isolated and characterized GFP from 10,000 jellyfish off the coast of WA in 1962 

•1990s: Chalfie constructed six individual nerve cells in C elegans to glow green 

•1990s: Tsien expanded the range of GFP-like proteins to express range of colors 

•-> Who constructed the gene?

“…missed it, by this much”


…and this much

•Stem cells

•http://en.wikipedia.org/wiki/Embryonic_stem_cell 

•http://en.wikipedia.org/wiki/Stem_cell 

Stem cells 

•1960s: EAMcCullock and JETill 

•Characterized by ability to replicate, renew themselves and differentiating into specialized cells 

•Two broad types: embryonic (found in blastocyts) and adult stem cells (found in adult tissues) 

•Pluripotent vs multipotent: 

multipotent found in adults that form limited number of cell types



Stem cells: “adult stem cells” 

•Pluripotent vs multipotent: 

multipotent found in adults that form limited number of cell types 

•Question of “adult stem cells”- 

•2005: Pluripotent stem cells from adult fibroblast cultures

•Question of “adult stem cells”- 



Stem cells 

•Stem cells discovered from analysis of teratocarcinoma (germ cell) in 1964 

•A single cell can be isolated and remain undifferentiated in culture, “embryonic carcinoma cell” 

•Cultured and stimulated to produce many different cell types 

•ES cells first derived from mouse embryos in 1981 (MEvans, MKaufman, GRMartin) 

•2005: Pluripotent stem cells from adult fibroblast cultures


Embryonic stem cells 

•Embryonic stem cells are stem cells derived from inner cell mass of early stage embryo, “blastcyst” 

•Human embryos reach blastocyst stage at 4-5 days; 50-150 cells 

•Pluripotent: into three primary germ layers, ectoderm, endoderm and mesoderm 

•Adult body has >220 differentiated cell types


•http://en.wikipedia.org/wiki/Stem_cell_treatments 

Embryonic stem cells 

•Plasticity and unlimited capacity for self-regeneration 

•ES cell therapies proposed for regenerative medicine and 

tissue replacement after injury or disease 

•2008: no approved treatment, yet from embryonic stem cell research 

•Adult stem cells and cord blood stems cells used to some diseases 

•Blood and immune-related genetic diseases, cancers and disorders 

•Juvenile diabetes 

•Parkinson’s 

•Blindness 

•Spinal cord injuries 

•NYT Aug06 

•Some scientists see shift in stem cell hopes 

•ie main role is in research 

•Cell therapy not as the first goal of the research

Irving Weissman: hemapoietic stem cells 

•http://findarticles.com/p/articles/mi_m1511/is_n3_v16/ai_16597943

•http://en.wikipedia.org/wiki/Stem_cell_treatments 

Embryonic stem cells

•http://en.wikipedia.org/wiki/Amgen 

The biotechnology of stem cells





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

The history of rbc production 

•1906 PCarnot proposed rbc prodution regulated by hormones 

•Carnot and DeFlandre studied rbc production in rabbits/bloodletting expts 

•“hemopoietin” and purified 

•1970s JAdamson and JWEschenback: sheep and other animals- EPO and renal failure 

and EPO stimulating rbc production in bone marrow; treatment of human anemia 

•1980s Adamson, Eschenback et al: clinical trial for synthetic form of EPO: Epogen 

•1985 Lin et al isolated h-erythropietin gene from genomic phage library 

•Gene encoded erythropoietin that is biologically active in vitro and in vivo 

•Production of recombinant erythropoietin (RhEpo) 

•1989 FDA approves Epogen 

•2000 novel erythropoiesis stimulating protein (NESP) produced; 

•Glycoprotein with anti-anemic capabilities and longer half-life 

•Offers effect at a lower dose


The biotechnology and use of rbc production 

•Therapeutic agent produced in mammalian cell culture 

•Treats anemia resulting from chronic kidney disease 

•From chemotherapy and radiation (cancer treatments) 

•Other critical illnesses, eg hear failure 

•But 

•Blood doping


The genomics of rbc production


Biochemistry of hematopoiesis


Bioinformatics of hematopoiesis

•http://en.wikipedia.org/wiki/Hematopoiesis 

Molecular biology of hematopoiesis 

•Formation of blood cellular components 

•All derived from hematopoietic stem cells 

•Stem cells in marrow; 

•unique ability to give rise to different blood cell types 

•10e11 to 10e12 new bood cells per day



•Self-renewing pools; some daughters remain HSCs as some differentiate 

•Lineages: 

•Erythroid: rbc 

•Lymphoid: immune cells 

•Myeloid: immune cells eg macrophages and blood clot functions



•Determination a function of molecular signal: cytokines 

•Maturation: gene expression pattern changes, differentiate 

•Can follow by cell surface markers 

•Determination: dictated by location of differentiation 

•Thymus for T-cells;


“simple” protein molecule

•http://en.wikipedia.org/wiki/Image:EPO.png 

Possibilities of the protein, the gene

Lecture 7: Human applications of the 

products of molecular biotechnology 

•Directed Mutagenesis (Chapt 8) 

•Protein engineering (Chapt 8) 

•Therapeutic agents (Chapt 10) 

•Monoclonal antibodies (Chapt 10) 

•Gene therapy (Chapt 10) 

•Molecular diagnostics

Lecture 6: Chapter 8 

•Directed mutagenesis 

•Protein engineering

Mutagenesis 

•In vivo 

•In vitro 

•Mutations occur spontaneously 

•Single base changes in the genome 

•Indels 

•Duplications 

•Mutations occur in response to ‘stimulus’ 

•Single base changes in the genome 

•Indels 

•Duplications 

•Mimic or expedite ‘naturally’ occurring mutations 

•Attractive given recombinant DNA technology 

•Overproduction and purification 

•“Make” better version 

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

Natural selection and 

growth: How? 

•Biological perspective of mutations 

•Glucose- depleted 

•Fructose- not available 

•Maltose- not available 

•Lactose- available

or in the presence of 

deleterious event 

•Population overgrowth -> colicin 

•[Antibiotic challenge -> ampicillin] 

•George Church, ISB 10/10/08 “natural and prvealent antibiotic resistance” 

•Environmental challenge -> radiation, heavy metals, ‘poisons’ 

•Natural selection, adaptive evolution 

•Whole organism, population versus molecular level

Natural mutation: HBB, beta hemoglobin gene 

•Molecular, genetic and biochemical perspectives 

•1600 bp, three exons 

•mRNA 626 bp; 44 bp codes for AAc 

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

•Left: hemoglobin, green and blue= alpha chains 

•Gold and aqua= beta chains 

•Gold spheres= phosphates; box= Glu6 

•Variant at 6= most common variant sickle cell “HBS” 

•Right: clumping of two hemoglobins with variant AAc 

•Several 100 HBB variants 

•Autosomal recessive 

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

Spontaneous chemical reactions 

•Chemical basis of mutations 

•Bases undergo random modifications 

•If uncorrected, mutation is hard copied 

•“SNP”

Another perspective: people and mutations 

•Essay, NYTimes. Oct 9, 2007. BHLerner, MD 

•Longevity reflects how the understanding of sickle cell disease has changed 

•Initially misdiagnosed in early 1960s- “found its way into the public consciousness.” 

•First complaints of joint aches- met with skepticism 

•At 16, diagnosed with 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-prescribe pain meds, requiring blood tests first

Modifying proteins 

•Mimicking natural mutations 

•Protocols to change specific amino acids encoded by a cloned gene 

•Therapeutic and industrial applications 

•Desired results: 

•Alter Michaelis constant Km- reflects substrate binding strength 

•Maximal rate of conversion Vmax 

•[improves efficiency of enzyme-catalyzed reaction Vmax/Km] 

•Change thermal tolerance, pH stability 

•Modify active site and structure in nonaqueous solvents, non-physiological conditions 

•Alter need for co-factor or change co-factor 

•Modify substrate-binding site to increase specificity, decrease side reactions 

•Link enzymes/proteins together 

•Increase resistance to proteases, extending half-life 

•Alter allosteric regulation events 

•“Addressing” /”export” issues of the protein 

•Purification issues 

•“Tracking” or visualization of protein

Site-directed mutagenesis 

•Theoretically, can modify protein at protein-level or gene-level 

•Protein-level: chemical, post-translational, modifications are harsh, nonspecific and tedious 

•But, mirrors Post-translational and epigenomic 

•Structure and predictive tools necessary for gene-level 

•“Directed mutagenesis” -generate amino acid coding changes at DNA level

Oligonucleotide-directed mutagenesis 

with M13 DNA 

•Best case scenario, let Nature do the work 

•Known sequence; available clone 

•Clone into ssM13 vector 

•Mismatch with mutation of desire 

•DNA Pol-based synthesis 

•Tf E. coli with ds recombinant vector 

•Expect 50%; in practice, 1-5% recovery 

•Why?

Tools: deamination repair systems

Mismatch repair: “simple”

Enrichment for mutations: 

dut/ung 

•Limited by imagination! 

•Use repair mutants of E. coli 

•1) dUTPase - (dut) 

•Elevated dUTP, some incorporated instead of dTTP 

•2) Uracil N-glycosylase - (ung) 

•Does not correct out U in DNA 

•Approx 1% U in place of T 

•Add oligo plus site-directed mutation 

•Generate dsDNA as before 

•Tf to ung + strain 

•Original strand is “repaired” ie degraded 

•Mutated strand is sustained

Variation: Directed 

mutagenesis with plasmid 

DNA 

•Drawback to M13-based system 

•Large number of time-consuming steps to isolated gene 

•Gets rid of need to subclone gene into M13 vector and 

subcloning mutated gene back out 

•Use MCS to clone in 

•Also, swap out Ap S /Tc R to Ap R /Tc S as selections

Variation: Directed 

mutagenesis with PCR 

•Faster protocol 

•Two-reaction protocol 

•Using ‘off-set’ primers 

•Synthesize each side of mutation 

•Pool, denature and renature, 

close gaps with ligase

Second generation variation: Random 

mutagenesis with 

degenerate oligonucleotide primers 

•Difficult to know which specific nuc to change 

•Especially in absence of structural data 

•Absence of functional data 

•Mass synthesis of mutants 

•Screen for ‘best desired’ 

•Can get unexpected mutants

Second generation variation: Random mutagenesis 

with degenerate oligonucleotide primers 

•Difficult to know which specific nuc to change 

•Especially in absence of structural data 

•Absence of functional data 

•Mass synthesis of mutants 

•Screen for ‘best desired’ 

•Can get unexpected mutants

Random mutagenesis with nucleotide analogs 

•Nucleotide analog resembles one of four bases 

•Use repair mechanisms of E. coli to incorporate

Random mutagenesis with 

error-prone PCR 

•Some DNA Pol have higher rates of misincorporations 

•Especially if remove proof-reading exo 

•Taq Pol 

•Mn ++ substitution

Third generation: DNA 

shuffling 

•Either 

•Random mutagenesis or 

•Error-prone PCR or 

•Shuffling large regions from different sources

Hybrids generated by 

shuffling protocol 

•Shuffling large regions from different sources 

•Use three RE sites 

•Two genes from same gene family

Hybrids generated by 

shuffling protocol 

•Shuffling large regions from different sources 

•Use three different RE sites 

•Several (3) genes from same gene family

Mutant proteins with non-universal amino acids 

•E. coli strain with novel tRNA system: 

•M. janaschii has tyr-tRNA synthase 

adds AAc to amber suppressor tRNA 

•amber= UAG 

•ex., tRNA for aminobutyrate

Insert into cell system that is appropriate 

•Subclone into appropriate vector 

•Transform cell type of interest 

•Overproduce 

•Characterize 

•Use 

•[Pretty routine….]

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”

Reasons to mutate: 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, this RNase 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- undesired 

•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

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

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 -> 174x thermal stability and 100x oxidative stability 

•Cherry…Pedersen. 99. Nat Biotech “Directed evolution of a fungal 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., 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 

•IFNα family of 13 genes; IFNβ family of 2 genes; IFNγ of 1 genes 

•Subtypes have different specificities 

•IFN α1 and α2 have similar antiviral activities when assessed with virus-challenged bovine cell line 

•IFN α2 is 7x more effective than α1 when human cells treated with virus 

•IFN α2 is 30x less effective than α1 when mouse cells treated with virus 

•IFN α1 and α2 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 

•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

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

Lecture 7 - Genome Tools

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