Biochemical Foundations of Pharmaceutical Biotechnology I

what is 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 rpecise sites into pieces

- cloning vectors (e.g. plasmids) to carry inserted "foreign" DNA fragments to produce more products (protein)

- how to make drugs from the beginning

- how to identify diseases and determine the treatment approaches

- how to personalize treatment modalities

how can you get therapeutic proteins from DNA sequences?

- proteins have been used to treat/prevent diseases in humans for over 100 years

- serum therapy for the treatment of tetanus and diphtheria

- antiserum obtained from immunized rabbits and horses

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

- eli lilly started large-scale purification of the pancreatic extracts to treat diabetes: 2 tons of pancreas from pigs/cows yielded 8 oz of insulin

- high degree of sequence conservation between pig/cow insulin to human insulin, made it possible to use in humans

what is the importance of the advancement of biotechnology?

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

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

what do biotechnologically derived drugs include?

- monoclonal antibodies

- endogenous or modified hormones

- growth factors

- antisense oligonucleotides

- vaccines

- DNA and RNA for gene therapy

- stem cell therapies

what are the differences between therapeutic proteins from classical, small molecule drugs?

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

- newly introduced biopharmaceuticals are expensive due to high cost in development and initial production costs

- high failure rates during drug discovery, the few that actually reach the market have to compensate for all the expenses from the failed ones

- the specificity of many therapeutic proteins means they are only effective in subgroups of patients: personalized medicine

what is trastuzumab (herceptin)?

approved for breast cancer patients with high expression levels of the Her2 receptor on the tumor cells (20% of breast cancer)

what are cetuximab and panitumumab used for?

- treatment of metastatic colorectal cancer patients

- targets the epidermal growth factor (EGF) receptor

- successful treatment depends on the presence of EGF receptor on the tumor cells and the absence of additional mutations in 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 from the EGF receptor and be nonresponsive to the monoclonal antibodies

what are the steps required to produce a therapeutic protein?

→ target selection

→ isolation mRNA from cells that express the gene of interest

→ reverse transcriptase reaction

→ mixture of cDNAs

→ amplify selected cDNA using PCR

→ cloning into suitable expression vector

→ foreign DNA to be inserted

→ ligation

→ recombinant DNA molecule

→ transformation of E. coli and selection for ampicillin resistance

→ quality check: sequence, orientation

→ transfection of mammalian cells

→ stable expression is obtained by selection for G418 resistance

→ selection of cells with the highest expression level

→ upscaling of cell culture

→ purification of the recombinant protein (downstream processing)

→ quality check

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.)

→ formulation

→ therapeutic protein suitable for human use: efficacy and safety have to be shown before market entrance

what is a chromosome?

single long molecule of DNA within which are the sequences of individual genes

what are deoxyribonucleotides?

DNA is composed of 4 kinds of deoxyribose nucleotides: adenine, cytosine, guanine, thymine

what is the basic structure of DNA?

phosphate, deoxyribose sugar, base

what are the components of DNA?

- phosphodiester bond connects the sugar of one nucleotide to the phosphate group of an adjacent nucleotide

- 2 strands of nucleotides are joined together by H bonds between complementary base pairs

- A always base pairs with T: weaker (2 H bonds)

- C base pairs with G: stronger (3 H bonds)

- sugars and phosphate form the DNA backbone

what is endonuclease?

hydrolyzes internal bonds within a polynucleotide chain

what is exonuclease?

hydrolyzes from the end of a polynucleotide chain

what is phosphatase?

hydrolyzes a terminal ester bond that links a phosphate (diphosphate/triphosphate) to a terminal nucleotide at either 5' or 3' end

what are restriction enzymes?

binds to, recognizes, and digests DNA within specific sequences of bases called recognition sequences or restriction sites

what are palindromes?

- recognition sequences

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

what are blunt ends?

- restriction endonucleases that cut at precisely opposite sites in the 2 strands of DNA generate blunt ends without overhangs

- e.g. SmaI

what are 5' overhangs?

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

what is a 3' overhang?

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

what is the occurrence probability of DNA sequences of increasing numbers of nucleotides?

1/4^n

what is a vector?

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

- e.g. plasmid DNA

what are plasminds?

- different types of plasmids exist naturally in bacteria

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

what is the process of cloning a DNA fragment?

→ vector/plasmid

→ DNA fragment joined with DNA ligase

→ recombinant DNA

→ replication of recombinant DNA within host cells

→ isolation, sequencing, and manipulation of purified DNA fragment

what is the key to cloning a DNA fragment?

link it to a vector/plasmid DNA molecule that can replicate within a host cell

what is the process of E. coli cloning?

→ plasmid vector with tetracycline-resistance gene and EcoRI

→ fragments joined with DNA ligase

→ E. coli cell transformed with recombinany plasmid

→ transformed cells plated onto medium containing tetracycline

→ only cells containing recombinant plasmid survive to produce resistant colony

what is the process of forming recombinant DNA?

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

→ the cuts produce DNA fragments with cohesive ends

→ H bonding of cohesive ends of DNA from bacterial plasmid by base pairing

→ joined fragments will form a linear/circular molecule: other combinations of fragments can occur

→ DNA ligase covalently attaches the backbones of the 2 DNA fragments, producing a molecule of recombinant DNA containing human and plasmid DNA

what is the process of annealing complementary extensions after cleavage by restriction enzyme BamH1?

→ BamH1 recognition site is cleaved

→ anneal

→ bacterial cells cannot grow and replicate in bacterial cells

what is antibiotic selection?

the most common selection process to identify recombinant bacteria

what are selective markers in the 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

- e.g. kanamycin, ampicillin, tetracycline, etc.

what is the action of streptomycin and other aminoglycosides?

inhibit initiation and cause misreading of mRNA (prokaryotes)

what is the action of tetracycline?

binds to the 30S subunit and inhibits binding of aminosoyl-tRNAs (prokaryotes)

what is the action of chloramphenol?

inhibits the peptidyl transferase activity of the 50S ribosomal subunit (prokaryotes)

what is the action of cycloheximide?

inhibits the peptidyl transferase activity of the 60S ribosomal subunit (eukaryotes)

what is the action of erythromycin?

binds to the 50S subunit and inhibits translocation (prokaryotes)

what is the action of puromycin?

causes premature chain termination by acting as an analog of aminoacyl-tRNA (prokaryotes and eukaryotes)

what are bacterial plasmid vectors?

- circular

- max insert size (kb): ~6-12

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

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

what are bacteriophage vectors?

- linear

- max insert size (kb): ~25

- application: cDNA, genomic and expression libraries

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

what are cosmids?

- circular

- max insert size (kb): ~35

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

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

what are bacterial artificial chromosomes?

- BAC, circular

- max insert size (kb): ~300

- application: genomic libraries, cloning large DNA fragments

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

what are yeast artificial chromosomes?

- YAC, circular

- max insert size (kb): 200-2000

- application: genomic libraries, cloning large DNA fragments

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

what are Ti vectors?

- circular

- max insert size (kb): varies depending on the type of Ti vector used

- application: 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

what is the genomic library?

- contains all of the genomic DNA of that organism

- the specific clones that carries the DNA sequences of interest must be identified, isolated, and characterized

what do restriction endonucleases do?

- digest the complete DNA of an organism

- each DNA fragment that has been restricted is inserted into a vector

what does creating a library mean?

the process of subdividing genomic DNA into clonable fragments and inserting them into vectors

what is the process of the construction of a genomic library?

→ human DNA cleaved with restriction enzyme

→ millions of genomic DNA fragments

→ plasmids cleaved with the same restriction enzyme as the human DNA

→ DNA fragment inserted into plasmids by DNA ligase

→ recombinant DNA molecules

→ introduction of plasmids into bacteria

→ genomic library containing all restriction fragments of human DNA

what is colony hybridization?

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

what is the process of the first step of cloning?

→ cleave vector and genomic DNA with BamHI.

→ mix cleaved vector fragment and genomic DNA fragments and ligate

→ possibly ligation products

→ transform ligation products into E. coli cells and spread onto master plate

→ on master plate with ampicillin, only the cells with circular plasmids grow

what is the process of colony hybridization?

→ nitrocellulose/nylon membrane

→ place membrane on top of agar containing separated bacterial colonies

→ allow original cells to recover

→ invert membrane and lay over new agar surface; allow new colonies to regenerate on top of filter

→ replica filter on new agar

→ remove membrane with attached colonies; soak in successive solutions

→ (a) denaturing, (b) neutralize, (c) wash, (d) dry/fix DNA

→ bacterial DNA fixed on membrane

→ (1) hybridize with labeled probe, (2) wash, (3) autoradiography

→ identify (+) colony and pick into liquid culture

→ grow in liquid culture

what is the process of probe hybridization to single-stranded DNA in cloned DNA?

→ disc is treated so that the DNA denatures (strands separate)

→ a single strand probe of complementary radioactive RNA/DNA is added

→ the strands are allowed to re-anneal

what is the process of 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 DNA to smaller fragments

→ DNA fragments are separated by agarose gel electrophoresis

→ to identify the fragment that contains the DNA of interest, a specific probe is used to hybridize to the DNA fragments

what is sanger chain-termination DNA sequencing?

- uses dideoxynucleotides to terminate DNA synthesis

- yields a series of DNA fragments whose sizes can be separated by electrophoresis

- the last base in each of the fragment is known, so we can determine the sequence of the DNA product

- product is the complement of the original DNA strand

what is the sanger method?

- new strands separated by electrophoresis

- sequence can be read from bands on autoradiograph and original template sequence deduced

- longest fragment ends with a ddG, so G must be the last base in the sequence

what is the process of automated DNA sequencing using fluorescent tags?

→ template of unknown sequence

→ DNA polymerase, 4 dNTPs, 4 ddNTPs

→ denature

→ dye-labeled segments of DNA, copied from template with unknown sequence

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

→ DNA migration

→ computer-generated result after bands migrate past detector

what is the polymerase chain reaction (PCR)?

- amplifies a region of DNA between 2 predetermined sites

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

- heat-stable DNA polymerase (e.g. Taq polymerase) comes from

what are oligonucleotides?

- complements to the predetermined sites in PCR

- serve as primers for the synthesis of copies of the DNA between the sites

where does heat-stable DNA polymerase come from?

- e.g. Taq polymerase

- from a bacterium that lives in hot springs and has heat-stable enzymatic activities

what is the process of PCR?

first cycle:

→ add excess primers to target sequence

→ heat to separate

→ cool

→ add heat-stable DNA polymerase

→ synthesize new DNA

second cycle:

→ heat to separate

→ cool

→ excess primers still present

→ heat-stable DNA polymerase still present

→ DNA synthesis continues

third cycle:

→ heat, anneal primers, extend

→ the short strands, representing the target sequence, are amplified exponentially

→ subsequent cycles

what PCR applications are there?

- amplification of rare DNA

- human genetic testing and disease diagnosis

- DNA cloning

- forensic DNA analysis

- study gene expression

- paternity testing and determining family relationships

- human remains identification

- diagnostic tests for disease-causing pathogens

what are the types of RNA produced in cells?

mRNAs, rRNAs, tRNAs, small RNAs (including miRNA)

what is the function of mRNA?

codes for proteins

what is the function of rRNA?

forms part of the structure of the ribosome and participates in protein synthesis

what is the function of tRNA?

used in protein synthesis as an adaptor between mRNA and amino acids

what is the functions of small RNAs?

used in splicing, transport of proteins, silencing mRNA and others

what is RNA processing?

- cap and tail added

- introns excised and exons spliced together

what is splicing?

the process of intron removal and exon joining

what are exons?

protein-coding regions that must bbe joined by removing introns

what are introns?

non-coding intervening sequences

what happens to eukaryotic mRNA?

modified, processed, and transported

what is alternative splicing?

generates different mRNAs from a single gene