..wHaT acTuaLly m I sTudyiNg...
1st lecture...was about protein structure..well as we already knew protein have 4 types of structure ..primarily - consist of amino acid, carboxyl group, nitrogen group, hydrogen atom and chiral carbon...two protein bind together with peptide bond and forming dihedral angle which known as torsion angles either psi / phi..da bond and angle are fairly invariant in the knowing da protein structure by Ramachandran blot...secondary - the present of alpha helix and beta sheet (parallel or anti-parallel)..tertiary - at this stage protein start to folding due to the structure of protein become tightly packed bcoz water was excluded from the interior..at this phase protein only can be stabilized mainly by hydrophobic forces but ionic interaction and hydrogen bond also play apart..protein folded forming motif which da protein r identical or unindentical more dense and form domain...quaternary - protein consist of more than one domain and becoming more complex...these domain basically bind together by disulphide bridges and were stabilised with the present of hydrogen bonding, hydrophobic effect,electrostatic interaction...
2nd lecture...we were learned about protein production for biotech...heterologous protein product by biological diversity, variety of post-translational modification and rotein expression which posibbly by bacteria, yeast, insect,and mammals or cell free system..there some critera for protein to be selected which are scale, cost, application, drug screening and therapeutic uses and antibody...it also include bacteria for expression which is commonly uses, more economical, require simpler expertises can be automated...the common bacteria used are; ecoli, pseudomonas, bacillus and rhodobacter spheroids...the challenges faced by doing all these are membrane protein is da key to processes n comprise da major drug, structural n functional very hard to produce n cytoplasmid n periplasmic volume is 30 X greater than membrane volume for typical cell..
rhodobacter - a strategy to produce membrane protein, advantage : organism can be engineered to provide coordinated synthesis of foreign membrane protein with synthesis of new membrane that can incorporated...
yeast expression system (similar ti bacteria) - transformation of yeast by integration into yeast genome, propagation as episome n as artificial chromosome..elements for transformation include ARS - autonomous replicating sequence, 2u - the 2 micron circle origin/ origin of replication, Cen - centromere..there are 3 types of yeast vector; integrative plasmid - introducing gene into yeast chromosome, centromeric plasmid - contain yeast centromere n low copy number , episomal plasmid - called 2 micron n have high copy vectors in yeast..advantages; - high yield n high productivity, chemically defined media, stable production strains, durability n lower protein production cost...product produces same as bacterial system ;- post translational modification, yeast have cellular organelles n intracelular n extracellular expression....
baculovirus expession system - infect primarily insect cell, insert up to ~20kb, save for human consumption, approved for therapeutic uses...suitable for production of eukaryotic protein n proper folding and post-translational modofication...vectors are commercially available..problem occur by baculovirus ; slow generation n protein processing not similar to human cell...
mammalian expression system - transformed of cell lines n resource...American Type Culture Collection (ATCC)..it saves the immortalised cell lines for long term production...but the cell lines will transfected with the expression construct n selected for recombinant production...common cell lines ;- chinese hamster ovary(CHO), green monkey kidney (COS), human embryonic kidney (HEM) n etc...probs occur : high cost, complicated technology n fear of potential animal viruses to human...
expression in transgenic plant - plant cultivation..since the conventional farming obtained low production..protein can target at specific stage of development...no fear for human pathogens good for pharmaceutical proteins...produce high value of protein..
transgenic animal - large scale production of eukaryotic protein..generation n characterization same like plant (need time n cost)..inheret advantage from eukaryotic protein processing..pronuclear micro injection of somatic transfer for mammary gland of cows, sheeps n goat also for transgenic chicken/egg of shorten life cycle...
cell free expression system - uses cell extract from various organisms containing ; transcription n protein components like cofactors n ATP cytosolic components...this system give advantage in terms of ; easily automated and scalable , flexibility for manipulation of condition for desired protein and possible for toxic protein..problem is the cost to develop the exact complexity of cellular system...
3rd lecture..methods for extraction protein...mechanical methods ; grinding, homogenizer or sonication or by using enzymatic method...da most common types for mechanical method are shear forces, homogenizer n french press...for enzymatic method is very gentle n specific means to disrupting the cells to release their contents..example for enzyme in used are lysozyme - bacteria cell wall, lyticase/chitinase - yeast n cellulose - plant...regularly osmotic shock been used by cytosolic outburst due to the entrance of water from out side cell into da cell...the second step is that clarification of extract...filtration n centrifugation ... third step is that protein purification...the purified must be possible in few steps n short a time..the target uses are therapeutic uses, industrial uses n structural uses...the sources of protein are eukaryotic n prokaryotic ( native; expressed constitutively by organism / recombinant; expressed from plasmid that has been transformed into host cell)..assay for protein include; colonrimetric/direct spectrophotometric - determine concentration, SDS-PAGE - determine homogeneity at each step n m.w of pure protein, enzyme assay- either disappearance of [S] / formation of [P], either one gives good spectral handle...
concentrating protein - important; low amount of enzyme, dilute form, extraction resulting loss of enzyme, easy to handle in small volume, easy assay at high concentration...concentration methods ; freeze drying - removal of water from a sample n keep under low temperature n under go sublimation , dialysis - diffusion of solutes through semi permeable membrane at different concentration of 2 solutes, salt precipitation - water soluble n their solubility as a function of ionic strength n pH of solution, ultrafiltration - pallet redissolved in low salt buffer since different protein have different distint characteristics.., etc...
purification - removes as much as contaminating protein as possible..caculate to now the efficency of the purification by specific activity X (% of yield / 100) ...protein characterization include molecular weight which can be determined by electrophoresis (SDS-PAGE), gel filtration n ultracentrifugation , isoelectric point ~ isoelectric focusing , spectroscopic propertieslike uv visible spectroscopy, absorbance spectroscopy, fluorescence spectroscopy, circular dichroism (CD) spectroscopy n NMR spectroscopy, determination of primary structure by inference n chemical method and lastly complete 3D structure by x-ray difraction n NMR...
