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63 Cards in this Set

  • Front
  • Back
Bacteriophage vectors vs. plasmid vectors (2)
1)bacteriophage can splice it's genome into bacterial chromosome
2)bacteriophage can hold 20kb of DNA (as opposed to 10kb)
Genetically engineered bacteriophages have...
2 BamHI sites in their genome
20kb of DNA can be removed from a genetically engineered bacteriophage w/o...
affecting its ability to infect a cell
Weaknesses of using a prokaryotic host cell for a vector (2) and why are these weaknesses HUGE?
1)Can't perform post translational modifications (like glycosylation)
2)Can't excise introns

These fxns are needed for biotech drugs that are glycoproteins which require glycosylation
Yeasts are used instead of E. coli sometimes b/c...(2) (and ex of a yeast used)
1)rapidly replicate
2)can perform post-translational modifications like glycosylation

S. cerevisiae
Yeasts as hosts for a vector weaknesses? (3)
1)expensive to maintain in culture
2)OVER glycosylate proteins
3)can't excise introns
Yeasts and prokaryotes both require ____ to be a vector
Use of higher eukaryote cells as vector hosts (adv(2) and 2 types of cells used
1)can remove introns
2)can do post translational modifications (glycosylations)

1)Chinese Hamster Ovary cells
2)Baby Hamster Kidney cells
Weakness of using eukaryote cells as vector hosts (3)
1)hard to maintain in culture
2)high cost
3)do not replicate very rapidly
Diabetes Type 1 (3)
1)pancreas does NOT make insulin
2)juvenile diabetes
3)insulin-dependent diabetes
Diabetes Type 2 (3)
1)usually happens via obesity
2)insulin independent
3)cells can't effectively bind insulin
Symptoms of DM2 (3)
2)"sweet" urine
3)excessive urination
Steps to make insulin (2)
1)Preproinsulin cleaved to proinsulin and signal peptide upon entering B cells
2)proinsulin cleaved to insulin, c-peptide, and dipeptides
____ guides preproinsulin to B cells
signal peptide
Insulin consists of...
an A and B chain
What joins A and B chain of insulin together
2 INTERdisulfide bonds
1 INTRAdisulfide bonds
Problems w/ porcine/bovine insulin
P-differed by 1 AA on B chain, so ppl got allergic rxns

B-differed by 2AAs on the A chain so allergic rxns
How is insulin stored in a B-cell
Stabilized by Zn it forms a hexamer
Insulin must be ____ to be active in the body
MUST BE A MONOMER, not the hexamer form stored in the B-cells
Gilbert's method of cloning the insulin gene (6)
1)preproinsulin cDNA isolated from induced tumors in rat pancreas
2)Add poly C tails to cDNA
3)Utilize PstI restriction site in ampicillin in pBR322
4)Add poly G tails to opened ampicillin resistance site
5)mix rat cDNA w/ pBR322 and T4 ligase
6)pBR322 vector w/ rat insulin gene is created
Problem w/ GILBERT's method
created hybrid protein (proinsulin/pencillinase), but was SECRETED
GILBERT's protein was proven to be secreted via...
Found it in periplasm via Ig method, shocking membrane releases hybrid protein from periplasm
Rutter and Goodman's method of cloning insulin gene (7)
1)Make cDNA w/ reversetranscriptase from mRNA of human insulin
2)Remove mRNA w/ alkali/RNAaseH
3)use reverse transcriptase to duplicate strand
4)cut "hairpin" w/ s1 nuclease
5)Add HindIII linkers by blunt-end ligation
6)Cut gene and pBR322 plasmid to produce sticky ends
7)mix gene, plasmid, and T4 ligase and vector is created
S1 nuclease...
ONLY attaches to one stranded DNA
Problem w/ Rutter/Goodman method
Genetech difference from Gilbert and Goodman?
didnt use cDNA just synthesized GENES for insulin A/B chains
Basic steps of Genetech's insulin cloning? (6)
1)A and B gene synthesized separately but all this is done to both genes
2)link gene to B-gal gene using a methionine
3)Insert gene (A or B and B-gal) into EcoRI site of pBR322, compromizing TCN resistance
4)Insert vector into E. coli and harvest protein
5)Split A/B from B-gal using cyanogen bromide (cuts the methionine connection)
6)Mix A/B chain together
1st insulin on the market was...
Human insulin (2 types and host cell used)
1)Humilin (E. coli)
2)Novolin (S. cerevisiae)
Lispro insulin
a)brand name
b)rate of axn
c)how different from human insulin
2)rapid acting (15-30min)
3)has a lys and pro switched on B chain
Aspart insulin
a)brand name
b)rate of axn
c)how different from human insulin
2)rapid acting (5-10min)
3)Asp where pro should be
Glargine insulin
a)brand name
b)rate of axn
c)how different from human insulin
b)ultra long lasting (24-48hr)
c)2 extra Arg on B and Gly where Asn is on A
Why is Lantus long lasting?
All AAs added are basic, they increase half-life due to precipitation in blood forming depots
How is insulin synthesized today? (2 and source)
1)mRNA to cDNA of proinsulin and put in E.coli
2)treat w/ enzymes to release pro part
3)soure mRNA got from pancreatic cancer
Clinical uses of HGH (5)
2)HGH deficiency in kids/adults
3)growth failure in kids due to renal insufficiency
4)Turner's Syndrome
5)Prader-Willi Syndrome
Commercially available recombinant HGH products (2)
a)generic name
b)company produced by
c)difference from human HGH
b)Eli Lilly
Most current formulations of rHGH are...
lyophilized (freeze dried) that are reconstituted prior to injection
Other dosage form of rHGH
2 different liquid formulations available
Why is HGH being used now more often in the elderly
to decr fat and incr lean
FSH is classified as... (2)
1)glycoprotein hormone
2)gonadotropin that acts @ male and female gonads
Clinical uses of rFSH (3)
1)stimulate spermatogeneis in men w/ hypogonadotropic hypogonadism
2)Ovarian hyperstimulation in women who are unresponsive to clomiphere
a)host cells used
a)chinese hamster ovary
2 rFSH products on the market
1)Folitropin alpha
2)Folitropin beta
Folitropin alpha brand names (2)
2)Seronois A
Folitropin beta brand names (2)
Coronary clots are formed from...(2)
1)aggregated platelet cells
2)insoluble fibrin which represents matrix of the clot
Thrombolytic agents effect on the clot (2)
1)bind to fibrin of clot
2)lead to fibrin degradation products which are soluble and hence lyse the clot
First recombinant human tPA activator
Alteplase (Activase)
tPA fxn (2)
1)conversion of plasminogen to active plasmin by hydrolysis of Arg560-Val561 peptide bond of the plasminogen
2)plasmin degrades the insoluble fibrin clot into soluble degradation product
tPA characteristics (5)
a)high affinity for fibrin
b)strong plasminogen activator
c)tPA enhances activation of plasminogen by 600x in presence of fibrin
e)less SEs than other thrombolytics b/c so clot specific
a)produced by... (2)
a)cDNA cloning technology
b)expressed in CHO cells b/c glycosylated protein
tPA is synthesized by...and properties of the chemical structure (5)
a)vascular endothelial cells

b)single chain protein
d)serine protease activity
e)17 disulfide bonds
f)Asp117 critical for its liver metabolism
tPA structural domains and which 2 bind fibrin (5)
1)finger domain***
2)growth factor domain
3)kringle domain1
4)kringle domain2***
5)serine protease domain
Clinical usage of Alteplase (rtPA) (3)
1)treat AMI
2)acute massive pulmonary embolism
3)ischemic stroke
Adverse rxns of alteplase (rtPA) (6)
1)bleeding (internal/superficial)
2)co-use of other anti-coagulants further incr bleeding
3)mild hypersensitivity rxns
2nd generation of recombinant thrombolytic agents are...(2)
1)analogues of alteplase
2)made b/c t1/2 of alteplase is less than 5min due to Asp117 n-glycosylation
Disadvantages of alteplace in AMI treatment (2)
1)rapid clearance by liver means you have to give it by infusion
2)complicated administration scheme
tPA analogues produced b/c... (3)
1)prolong half-life allowing a single bolus admin
2)higher level of fibrin specificity to decr bleeding
3)resistance to endogenous tPA inactivtor
2 alteplase analogs
Reteplase difference from alteplase (5)
1)nonglycosylated deletion mutation
2)missing kringle1, finger, growth factor domain
3)expressed in E.coli b/c its non-glycosylated
4)longer half-life
5)greater thrombolytic POTENCY
Clinical usage of Reteplase (3)
1)imporved ventricular fxn following an AMI
2)reduction of CHF
3)decr mortality from AMI
Alteplase ADR's