->Proteases

Trypsin
-di-peptidase activity (not the word he uses, only Sadeem uses)
-normal activity
-recognise 2 +ve charge a.a (one after the other)
 -Arg, Lys, his (rare) out of the 2 a.a.
 -cleave after 2nd a.a. (C end; just cleaves there, not clip off)
 -ex. Trypsin like pancreatic enzyme for processing of natural insulin

-Other modified activity (excess trypsin present) -> have this modified activity when trypsin is in excess.
 -recognize K or R (not 2)
 -cleave after either K or R
 -ex. human-like procaine insulin

-modified activity (excess trypsin)
 -recognize K only
 -cut after "K" only (not R)
 -ex. Novo-Nard recombinant insulin

-Transpeptidation (modified activity when excess trypsin + transpeptides)
 -cleaves at target then add the transpeptide
 -ex: - human like procaine insulin
      - novo-Nard recombinant insulin

Kex 2 (protease of Saccharomycins in alpha-mating pathway
-very specific substrate: N-KR-C
-very specific activity - cleaves after R only
-efficient activity requires a N-terminus spacer (=cut at each and other sequence??)
-native spacer: EDEDEA (-ve charge sequence)
-recombinant spacer: EAEAEA = uncharged -> still negatively charged
   ->when for recombination

Dipeptidyl amino-peptidase
-protease of Saccharomyces - mating factor secreation
-clips off specific dipeptides (ED or EA)
   -> not cut after, clip off!!
-from N-terminus of alpha matin factor (alpha-MF) precursors (only happens if it is right at N-terminus (exoprotease)
-efficiency is reduced for recombinant protein
   ->not cutting every time, some have EA or ED left, some?? when produce recombinant insulin, too fast, this?? protease can't catch up to cut every product

Carboxypeptidase
-clips off a.a. from C-terminus
-some are specific to +ve charged a.a.
  -> some but all clip a.a. from C-ter
-ex. Kex 1
 - a protease of Saccharomyces of alpha-MF secretion, clips off +ve KR from (-form?? of alpha-MF precursors
 - pancreatic carboxypeptidase

Natural insulin (body produce)
-pancreatic cells, secreted to blood, go thru some phases before secreted (preproinsulin, proinsulin, mature insulin, insulin hexamer, secrete)
   -> all post-translational modification after pre-proinsulin

-structure of pre-proinsulin -> have signal leader 23 a.a.

MICB 421
Week 7 Participation Report

Team: 3 Zeta
Members:  Catherine Chan
 Timothy Chiu
 Ray Kang
Week: March 9-15, 2006 (Thu„³Wed)

Day Name Assigned Task Estimated time

Thu Mar. 2
9:30 am Catherine Monitor incubated plates and count colonies 15 min

 Timothy Collect flasks for autoclave
  Collect materials needed for experiments 15 min

 
1:00 pm Catherine  Prepare materials for PCR; perform PCR 1 hour
 Timothy
 
 Ray Prepare two agarose gels. One for checking      1 hour
  PCR products, the other one for checking
  ligation products  

Fri Mar. 3
12 pm Ray Obtain PCR results   30 min
 Timothy Ensure PCR product to be OmpA gene by
  performing restriction endonuclease digestion
  Run the fragments in gel electrophoresis
  
2pm Catherine Obtain results from gel electrophoresis.  30 min
  Confirm the gene amplified by PCR.

Mon Mar. 6
12 pm Catherine Perform ligation reaction using TOPO 2 hours
 Ray
 Timothy

Tue Mar. 7
10:00 am Ray Determine if PCR products are ligated by  1 hour
  performing gel electrophoresis

1:00 pm Timothy Transform recombinant plasmid into recipient  45 min
 Catherine cells
  Plate recipient cells to confirm transformation
 
Wed Mar. 8
12 pm Timothy Monitor increased level of OmpA by Western  2 hours
 Ray blot
 Catherine