in-vitro mutagenesis

what are the differences between NMEs and BLAs?

differences in size and complexity of small molecule drugs vs. recombinant factor rFVIII or monoclonal antibody by molecular modeling

what are the key concepts of protein engineering?

• design and construction of proteins by recombinant DNA techniques

• native proteins are not well suited for industrial application

• native proteins are not optimized for medicinal purposes

what are biologics?

• generally large, complex molecules

• produced from a living organism

• used in the prevention, diagnosis, or treatment of diseases

what are biological drugs?

include antibodies, growth hormones, interleukins, and vaccines

what is possible with recombinant DNA technology?

• isolate the gene for any protein that exists in nature

• express it in a specific host organism

• produce purified products that can be used commercially

what is the human growth hormone?

191 amino acid, 22 kDa

what is the feedback regulation of human growth hormones?

• IGF-1 inhibits AP and GHRH

• IGF-1 stimulates somatostatin, bone growth, muscle growth

• somatostatin inhibits AP

• ghrelin stimulates AP and GHRH

• AP stimulates GH (negative feedback loop)

• GH stimulates liver: release of IGF-1

what is pediatric growth hormone deficiency?

• disease in which a child does not have enough growth hormones

• recombinant human growth hormone is used to treat short stature due to pGHD, turner syndrome, and idiopathic short stature

what is recombinant human growth hormone "somatropin"?

• used for pGHD with a well-established efficacy and safety profile

• same sequence 191 amino acid, 22 Kda, as natural growth hormone

• administered once daily as a subcutaneous injection before bedtime

• many children with pGHD fail to achieve their target adult height

what are the compliance issues with somatropin?

70% non-adherence due to long-term administration

what is recombinant albutropin?

fusion protein that includes human serum albumin at the N terminus and human growth hormone at the C terminus

what are strategies for long-acting growth hormone?

formulation, changes in hGH

how is formulation used for long-acting hGH?

modify the formulation of the GH to allow the product to be slowly released from the injection site over a period of time (no change in the active ingredient)

how are changes in hGH used for long-acting hGH?

modify the GH molecule itself so that the active drug is cleared more slowly from the systemic circulation

what is somatrogon (ngelna)?

• glycosylated

• comprises the amino acid sequence of hGH and one copy of the C-terminal peptide (CTP) from the beta chain of human chorionic gonadotropin (hCG) at the N-terminus and 2 copies of CTP (in tandem) at the C-terminus

• size: 40 Kda

• glycosylation and CTP domains account for the half-life of the molecule

• once weekly subcutaneous injection

• compliance issues with prior somatropin is improved

• FDA approved for pGHD: once weekly subcutaneous injection

what is somapacitan-beco (sogroya)?

• for adults with growth hormone deficiency

• a human growth hormone (hGH) analog with a single substitution in the amino acid backbone (leucine 10 cysteine) to which an albumin-binding moiety has been attached

• somapacitan bound to albumin (delayed elimination)

what is DNA biotechnology?

by using a set of techniques that change specific amino acids encoded by a cloned gene, proteins with properties that are better suited than naturally occurring counterparts can be created for therapeutic and industrial applications

what is site-directed mutagenesis?

• oligonucleotide-directed mutagenesis

• single-stranded oligonucleotide, which can be routinely synthesized by chemical methods, is used for generating point mutations

what is oligonucleotide-directed mutagenesis using M13?

• site-specific mutagenesis to make a point mutation in a cloned gene: must know the precise nucleotide sequence in DNA that encodes the mRNA

• single-stranded bacteriophage M13 (M13+ strand), carrying a cloned gene, is annealed with a complementary synthetic oligonucleotide containing a mismatched base

what are the stages of M13 phage DNA replication?

1. the infecting single-stranded circular DNA (+ strand) is replicated

• a complementary strand (- strand) is synthesized

• conversion of the infecting, single-stranded, circular DNA in a double-stranded replicative form of DNA

2. semi-conservative replication

• the number of double-stranded replicative-form DNAincreases to 100-200 copies per cell

3. asymmetric replication

• after the number of double-stranded replicative-form DNA has increased, only the + strand is produced and incorporated into virus particles

what is the process of oligonucleotide-directed mutagenesis using M13?

1. a single-stranded M13 phage DNA contains a gene of interest

2. mutagenic oligonucleotide annealing

• a short synthetic oligonucleotide is designed to be mostly complementary to the template, except for one mismatched base

• the mismatch introduces the desired mutation

3. second-strand synthesis

• the oligonucleotide provides a 5' phosphate and anneals to the M13+ strand

• klenow fragment (DNA polymerase I) plus dNTPs extends the primer, copying the rest of the strand while incorporating the mutation

4. T4 DNA ligase seals the remaining nicks, producing a double-stranded, closed, circular DNA molecule containing one wild-type and one mutant strand

5. DNA replication in E. coli

• strand segregate, yielding wild-type and mutant M13 phage

what is M13?

• filamentous bacteriophage (bacteria virus) which infects E. coli host

• has closed circular DNA genome

what is oligonucleotide-directed mutagenesis using plasmid DNA?

• no M13 bacteriophage system

• the target DNA is inserted into a multiple cloning site (MCS) on a plasmid vector that contains a functional tetracycline resistance gene and a non-functional ampicillin resistance gene

what is the process of oligonucleotide-directed mutagenesis with plasmid DNA?

1. starting plasmid contains:

• a multiple cloning site where DNA can be inserted: lies within a selectable region, so changes can be detected

• an ampicillin resistance gene

• a tetracycline resistance gene

2. the target DNA fragment is inserted into the plasmid at the MCS using restriction enzymes and ligation, producing a recombinant plasmid carrying the gene of interest

3. a specific mutation is introduced into the target DNA sequence

4. the plasmid now contains mutated target DNA

5. antibiotic resistance pattern changes

• amp: cells carrying the plasmid survive on ampicillin

• tet: tetracycline resistance is disrupted due to insertion/mutation

6. selection and screening

what is the process of plasmid-based site-directed mutagenesis method?

1. starting plasmid vector contains:

• a multiple cloning site (MCS)

• an ampicillin sensitivity gene (Ampˢ)

• a tetracycline resistance gene (Tetʳ)

2. target DNA is cloned into the plasmid at the MCS

• the recombinant plasmid is propagated in E. coli and plasmid DNA is isolated

3. the plasmid DNA is treated with alkali, producing single-stranded circular DNA

4. short synthetic oligonucleotides are added that introduce a specific mutation in the target gene

5. introduce changes in selectable markers:

• an Ampʳ oligo (confers ampicillin resistance)

• a Tetˢ oligo (disrupts tetracycline resistance)

• these oligos anneal with intentional mismatches

6. T4 DNA polymerase extends from the annealed oligos

• DNA ligase seals nicks, forming a double-stranded plasmid containing the mutations

7. the plasmid is transformed into E. coli.

8. election identifies bacteria with the mutated plasmid:

• Ampʳ → cells survive on ampicillin

• Tetˢ → loss of tetracycline resistance confirms successful mutagenesis

9. final product: plasmid carrying the mutated target DNA and the desired antibiotic resistance pattern (Ampʳ Tetˢ)

what is direct mutagenesis using PCR?

• the gene to be mutated is cloned, and the entire sequence is known

• to alter one specific nucleotide, normal and mutagenic primers are combined in a PCR reaction

• the mutagenic primer will have a mismatch in the middle, but the remaining sequences will be complementary

what is random mutagenesis?

• error-prone PCR

• single amino acid substitutions

what is the process of random mutagenesis with nucleotide analogs?

1. the gene of interest is cloned into a plasmid

• 2 restriction enzyme sites (RE1 and RE2) flank the region to be mutated

2. the plasmid is cut with two different restriction enzymes, creating asymmetric ends:

• one 3′ recessed end

• one 5′ recessed end

3. exonuclease III (ExoIII) digests DNA only from a 3′ recessed end

• it removes nucleotides in the 3′ → 5′ direction on one strand only

• result: a single-stranded gap is created across part of the target gene

4. DNA polymerase fills in the gap using:

• 4 normal dNTPs

• 1 dNTP analog (e.g., 8-oxo-dGTP): incorporated at a specific position during synthesis

5. S1 nuclease removes any remaining single-stranded regions

6. DNA ligase seals the nicks, restoring a closed circular plasmid that contains the incorporated nucleotide analog

7. the plasmid is transformed into E. coli

• during replication, the nucleotide analog mispairs, leading to a permanent base-pair mutation in the plasmid DNA

what is error-prone PCR process?

• yields a variety of mutated forms of the gene

• PCR using DNA polymerase lacks a proofreading activity

• low fidelity

what is proofreading activity?

3' ---> 5' exonuclease activity

what are the uses of different DNA polymerases in mutagenesis?

• basic reaction catalyzed by DNA polymerase: newly synthesized strand

• bacterial DNA polymerase I: DNA polymerization + DNA degradation (existing nucleotides are replaced)

• the Klenow fragment: DNA polymerase I lacks 5' ---> 3' exonuclease activity (existing nucleotides are not replaces and only the nick is filled in)

• low fidelity DNA polymerase: lacks 3' ---> 5' exonuclease proofreading activity

what is DNA shuffling?

• recombination to produce hybrid or chimeric proteins

• works well with gene families (molecular breeding)

what is random mutagenesis with DNA shuffling?

• DNA shuffling mostly uses related genes to produce hybrid/chimeric proteins

• portions of similar genes are shuffled using same restriction enzyme sites

what is the concept of recombinant drugs?

• alteration of enzyme catalytic activity, specificity, and allosteric regulation

• alteration of thermal tolerance or pH stability

• increased resistance to proteases for better purification and better recovery rate

• modifying metal cofactor requirements

what is adding disulfide bonds in recombinant drugs?

• increases the thermostability of proteins

• use of oligonucleotide-directed mutagenesis

• changes in amino acid residues to cysteine (encoded by UGC or UGU)

• no changes in the active site of the enzyme

what is recombinant streptokinase?

• increased resistance to proteases

• blood-clot dissolving agent for heart attack, pulmonary embolism

• forms complex with plasminogen, resulting in converting plasminogen to plasmin: the active protease that degrades fibrin in the blood clot

• by converting plasminogen to plasmin, streptokinase is quickly degraded by plasmin

• protease cleavage sites lysine 59 and lysine 386 are changed to glutamine: protease resistant

what is plasmin?

a protease that specifically cleaves the peptide bond after a lysine/arginine residue

what is glutamine?

has a similar length of side chain and does not have a (+) charge, therefore not degraded by protease

what processed size of recombinant streptokinase has high activity?

47 kDa

what is the thermostability of recombinant drugs?

• at high temperatures, asparagine and glutamine residues undergo deamidation, releasing ammonia

• with the loss of the amide moiety, these amino acids become aspartic acid and glutamic acid, resulting in changes in folding of the peptide chain that may lead to a loss of activity

• engineering by changing asparagine to other amino acids

what is betaseron (interferon beta-1b)?

• first disease-modifying therapy approved to treat RRMS

• manufactured in E. coli that bears a genetically engineered plasmid containing the gene for human interferon beta ser17

• the native gene was obtained from human fibroblasts and altered to substitute serine for the cysteine residue found at position 17

what is inteferon-β?

• reduction of free sulfhydryl residues: synthetic interferon-β expressed forms inactive dimers/oligomers in E. coli

• intermolecular disulfide bonding: forming inactive dimers/oligomers

• intramolecular disulfide bonding: important for thermal stability

• solution: serine (containing O) replacing cysteine (containing S) = no multimeric complex formed in E. coli

what is the modifying metal cofactor requirement of recombinant drugs?

• under industrial settings using a large number of metal-chelating agents, removing Ca2+, it is rapidly inactivated

• need for increasing the stability of the enzyme

what is the genetic engineering of Ca2+-independent subtilisin?

• engineered subtilisin (maintained overall conformation, but no activity)

• involves a deleted region

what is molecular breeding in recombinant drugs?

• altering multiple properties

• portions of genes are recombined by DNA shuffling to produce a large # of new proteins

what is the process of molecular breeding?

→ screening and testing a shuffled DNA library of the subtilisin gene family
→ the entire library plated on agar plates with 2% milk
→ pick colonies that form a zone of clearance (digested milk proteins)
→ the chosen colonies grown in 96-well plates
→ add a chromogenic substrate to determine enzyme activity
→ assay activity under different conditions