Biochemical Foundations of Pharmaceutical Biotechnology (L16)

molecular biology

-development of tools and methods to directly manipulate DNA in vitro and in vivo

-restriction endonucleases allow DNA to be cut at the precise sites into pieces

-cloning vectors, like plasmids, carry inserted "foreign" DNA fragments to produce more products (proteins)

-how to make drugs from the beginning

-how to identify diseases and determine the treatment approaches

-how to personalize treatment modalities

molecular biotechnology from DNA sequence to therapeutic protein

-proteins are used to treat or prevent diseases in humans

-serum therapy is used for the treatment of tetanus and diphtheria, the antiserum was obtained from immunized rabbits and horses

-isolated and purified insulin from pig/cow pancreas to treat T1DM in the early 1920s

-high levels of sequence conservation between pig/cow insulin to human insulin made it possible to use on humans

the advancement of biotechnology

-made it possible to move almost entirely away from animal-derived proteins to proteins with amino acid sequences

-recombinant proteins are less likely to cause side effects and to elicit immune responses

-the share of biotechnologically derived drugs has increased over the last 30 years

therapeutic proteins vs classical, small molecule drugs

-differ in size, composition, production, purification, contamination, side effects, stability, formulation, and regulation

-therapeutic proteins are specific --> only effective in subgroups of patients (personalized medicine)

What does the successful treatment using cetuximab or panitumumab depend on?

1. the presence of EGF receptors of tumor cells

2. the absence of additional mutations in the downstream EGF receptor signaling effector molecules

What do mutations in one or more of the downstream EGF receptor signaling effector molecules cause?

tumor cells to grow independently form the EGF receptor and nonresponsive to the monoclonal antibodies

steps required to produce a therapeutic protein

1. target selection

2. isolation of mRNA from cells that express the gene of interest

3. mixture of cDNAs

4. amplify selected cDNA using PCR

5. cloning into a suitable expression vector

6. transformation of E.coli and selection for ampicillin resistance

7. quality check: sequence, orientation

8. transfection of mammalian cells

9. stable expression is obtained by selection for G418 resistance

10. selection of cells with the highest expression level

11. upscaling of cell culture

12. purification of the recombinant protein

13. quality check

14. formulation

15. therapeutic protein is suitable for human use, and evaluate safety and efficacy

quality check before the formulation of the therapeutic protein

1. process validation: removal of host DNA and proteins, viral clearance, etc.

2. consistency of the manufacturing process

3. consistency of the drug substance (glycosylation, folding, etc.)

average atomic mass of one base pair

-635 daltons

-a dalton is 1/12 the mass of a carbon mass

β-globin gene

-2000 bp in length

-atomic mass is 1.27x10^6 daltons

mass of the β globin in adult humans

-2 copies of β globin gene per cell, 10^13 cells per individual

-2.54x10^19 daltons

β globin DNA in an adult human in grams

-6.02x10^23 daltons per gram

-0.000042 g = 0.042 mg = 42 mcg

mass of the β globin in a liter of E. coli

-500 copies of β globin per cell, 5x10^11 cells per liter

-3.175x10^20 daltons

β globin DNA in a 1-liter culture of E. coli in grams

-6.02x10^23 daltons per gram

-0.000527 = 0.527 mg = 527 mcg

chromosomes

each chromosome has a single long molecule of DNA with many sequences of individual genes

phosphodiester bonds

connects the sugar of one nucleotide to the phosphate group of an adjacent nucleotide

hydrogen bonds

-join two strands of nucleotides together between complementary base pairs

-A-T

-C-G

endonuclease

hydrolyze internal bonds within a polynucleotide chain

exonuclease

hydrolyze from the end of a polynucleotide chain

phosphatase

hydrolyze a terminal ester bond that links a phosphate to a terminal nucleotide at either the 5' or 3' end

restriction enzyme

binds to, recognizes, and digests DNA within specific sequences of bases: recognition sequences/restriction sites

recognition sequences

palindromes: the arrangement of nucleotides reads the same forward and backward on opposite strands of the DNA molecules

Eco RI

-restriction endonuclease that cuts DNA at GAATTC

-source: E. coli R13

-recognizes 6 base pairs

-produces 4-base-long sticky ends

Hha I

-restriction endonuclease that cuts DNA at CGCG

-source: haemophilus haemolyticus

-recognizes 4 base pairs

-produces 2-base-long sticky ends

Sma I

-restriction endonuclease that cuts DNA at CCC-GGG

-source: serratia marcescens

-recognizes 6 base pairs

-produces blunt ends

Hae III

-cuts DNA completely in half between every GG-CC sequence

-source: haemophilus aegyptius

-recognizes 4 base pairs

-produces blunt ends

blunts

-restriction endonucleases that cut at precisely opposite sites in the two strands of DNA generate blunt ends without overhangs

-Sma I and Hae III

5' overhangs

-the enzyme cuts asymmetrically within the recognition site such that a short single-stranded segment extends from the 5' ends

-Bam H1, Eco R1

3' overhangs

-asymmetrical cutting within the recognition site, resulting in a single-stranded overhang from the two 3' ends

-Kpn 1

nucleotides and their occurrence probability (1/n)

-1 - 1/4

-2 - 1/16

-3 - 1/64

-4 - 1/256

-5 - 1/1,024

-6 - 1/4,096

-7 - 1/16,384

-8 - 1/65,536

-9 - 1/262,144

-10 - 1/1,048,304

vector

-DNA that can accept, carry, and replicate other pieces of DNA in molecular biology cloning experiments

-ex: plasmid DNA

plasmids

-different types of plasmids exist naturally in bacteria

-primary function is to provide bacteria with resistance to antibiotics, done through a protein/enzyme that will inactivate the function of a given antibiotic

cloning sequence

1. restriction enzyme cuts double-stranded DNA at its particular recognition sequence

2. the cuts produce DNA fragments with cohesive ends

3. when two fragments of DNA cut by the same restriction enzyme come together, they can join by base pairing

4. the joined fragments usually form either a linear or circular molecule; other combinations of fragments can also occur

5. the enzyme DNA ligase is used to untie the backbones of the two DNA fragments, producing a molecule of recombinant DNA containing human and plasmid DNA

annealing

recombine (DNA) in the double-stranded form following cleavage by the restriction enzyme BamH1

antibiotic selection

-the most common selection process to identify recombinant bacteria

-distinguishing between recombinant bacteria and ones that have not taken up the recombinant plasmids

selective markers in plasmids

-provide resistance to compounds in the growth media

-allow rare populations of bacteria that have taken up the incoming recombinant plasmids to grow while preventing the growth of bacteria that have not taken up recombinant plasmids with a piece of foreign DNA

-kanamycin, ampicillin, tetracycline

antibiotic inhibitors of protein synthesis

1. streptomycin and other aminoglycosides

2. tetracyclin

3. chloramphenicol

4. cycloheximide

5. erythromycin

6. puromycin

streptomycin and other aminoglycosides

-inhibit initiation and cause misreading of mRNA

-prokaryotes

tetracyclin

-binds to the 30S subunit and inhibits the binding of aminoacyl-tRNAs

-prokaryotes

chloramphenicol

-inhibits the peptidyl transferase activity of the 50S ribosomal subunit

-prokaryotes

cycloheximide

-inhibits the peptidyl transferase activity of the 60S ribosomal subunit

-eukaryotes

erythromycin

-binds to the 50S subunit and inhibits translocation

-prokaryotes

puromycin

-causes premature chain termination by acting as an analog of aminoacyl-tRNA

-prokaryotes and eukaryotes

DNA vectors

1. bacterial plasmid vectors (circular)

2. bacteriophage vectors (linear)

3. cosmid (circular)

4. bacterial artificial chromosome (BAC, circular)

5. yeast artificial chromosome (YAC, circular)

6. Ti vector (circular)

bacterial plasmid vectors

-circular

-maximum insert size: ~6-12 kb

-applications: DNA cloning, protein expression, subcloning, direct sequencing of insert

-limitations: restricted insert size, limited expression of proteins, copy number proteins, replication restricted to bacteria

bacteriophage vectors

-linear

-maximum insert size: ~25 kb

-applications: cDNA, genomic and expression libraries

-limitations: packaging limits, DNA insert size, host replication problems

cosmid

-circular

-maximum insert size: ~35 kb

-applications: cDNA and genomic libraries, cloning large DNA fragments

-limitations: phage packaging restrictions, not ideal for protein expression, cannot be replicated in mammalian cells

bacterial artificial chromosome (BAC)

-circular

-maximum insert size: ~300 kb

-applications: genomic libraries, cloning large DNA fragments

-limitations: replication restricted to bacteria, cannot be used for protein expression

yeast artificial chromosome (YAC)

-circular

-maximum insert size: 200-2000 kb

-applications: genomic libraries, cloning large DNA fragments

-limitations: must be grown in yeast, cannot be used in bacteria

Ti vector

-circular

-maximum insert size: varies depending on the type of Ti vector used

-applications: gene transfer in plants

-limitations: limited to use in plant cells only, number of restriction sites randomly distributed, large size of vector not easily manipulated

genomic library

-complete DNA of an organism is digested with a restriction enzyme

-each DNA fragment is inserted into a vector that has been restricted with the same endonuclease

-contains all of the genomic DNA of an organism

-created by subdividing genomic DNA into clonable fragments and inserting them into vectors

construction of a genomic library

1. cleave human DNA with a restriction enzyme --> millions of genomic DNA fragments

2. plasmids cleaved with the same restriction enzymes as human DNA --> DNA ligase inserts the DNA fragments into plasmids

3. introduce plasmids into bacteria --> genomic library containing all restriction fragments of human DNA

colony hybridization

library screening with a DNA probe to identify a cloned gene of interest

process of colony hybridization

1. replicate colonies on a positively-charged membrane

2. isolate DNA, label with radioactive phosphate

3. view on x-ray to identify the positive colony

4. pick the positive colony into liquid culture

5. finished product: clone of interest identified and grown into liquid culture

probe hybridization to single-stranded DNA in cloned DNA

1. Disc is treated so that the DNA denatures (strands separate)

2. a single-stranded probe of complementary radioactive RNA or DNA is added

3. the strands are allowed to re-anneal

DNA hybridization

-the presence of a specific gene has to be identified before it can be used for further analysis

-restriction enzymes are used to reduce the DNA into smaller fragments

-DNA fragments are separated by agarose gel electrophoresis

-a specific probe is used to hybridize the DNA fragments in order to identify the fragment that contains the DNA of interest

Sanger Chain-Termination DNA Sequencing

-uses dideoxynucleotides to terminate DNA synthesis --> series of DNA fragments with sizes separated by electrophoresis

-the last base in each fragment is known since we know which dideoxy nucleotide was used to terminate each reaction

-ordering the fragments by size from the bottom of the gel

-each fragment is one base longer than the previous one

-the product is the complement of the original DNA strand

dideoxynucleotides

-a nucleotide with an H at C3 instead of an OH

-prevents the formation of a phosphodiester bond to the next deoxynucleotide

-prevents the backbone from elongating and terminates it at that point

automated DNA sequencing using fluorescent tags

1. template of unknown sequence --> DNA polymerase, 4 dNTPs, 4 ddNTPs

2. denature --> dye-labeled segments of DNA copied from the template with an unknown sequence

3. dye-labeled segments are applied to a capillary gel and subjected to electrophoresis

PCR (polymerase chain reaction)

-amplifies a region of DNA between two predetermined sites

-oligonucleotides complementary to these sites serve as primers for the synthesis of copies of the DNA between the sites

-each PCR cycle doubles the number of copies of the amplified DNA

-heat-stable DNA polymerase (Taq) comes from a bacterium that lives in hot springs

PCR sequence

1. denaturing dsDNA at 94°C

2. annealing primers at a temperature 5°C below Tm

3. synthesizing new DNA at 72°C

4. cycle repeats 25-30 times

PCR applications

-human genetic testing and disease diagnosis

-DNA cloning

-studying gene expression

-diagnostic tests for disease-causing pathogens

-human remains identification

-paternity testing and determining family relationships

-forensic DNA analysis

-amplification of rare DNA

T/F: Restriction enzymes cut RNA at precise locations

False

1 multiple choice option

T/F: One can predict how many times a specific restriction enzyme may cut a precise fragment

True

T/F: Antibiotic selection in recombinant DNA cloning is one way to ensure that the desired "foreign" DNA is inserted in the vector

True

1 multiple choice option

T/F: The incorporation of a dideoxyribonucleotide terminates the elongation of the DNA strand

True

1 multiple choice option

T/F: PCR is a way to synthesize protein

False