Class Notes (806,766)
Canada (492,430)
Biochemistry (382)
BCH210H1 (352)

bch210 lecture7-9.docx

5 Pages
Unlock Document

University of Toronto St. George
Charles Deber

Lectures 7 to 9 Genetic engineering basic approach  Clone the gene of interest  Insert the gene in an expression vector  Insert the plasmid into a bacterial host  Grow the bacteria: they express the protein Site specific mutagenesis  Cleave out protein of interest using restriction endonuclease sites  Add an oligonucleotide (has the mutation in the gene you want expressed)  DNA ligase the plasmid together  Transformation into bacteria  Alternating the gene is straightforward if the organism normally secrets the protein, if not the organism will be fooled into expressing the foreign protein during its growth cycle Restriction sites  Restriction endonucleases are enzymes found in prokaryotic cells  Function: degrade foreign DNA  They recognize palindromic sites in double stranded DNA (same backwards as forwards) – cleave phosphodiester bonds Synthetic oligonucleotides and its Inserition  Creating an oligonucleotide is adding nucleotides one by one on a resin support. You can change one base that will change a AA in the protein sequence.  Chemical synteheis oligos is performed on solid resin supports; oligos are cleaved (by endonucleases?) from the resin at the end of synthesis  Coupled reactions tend to be 100% complete in most cases. Gene machines have made this automated  Many genes contain wild type restriction sites: you just choose the right now to cut out your gene  Cleave the gene of interest at the restriction sites  Synthesize and purify the desired oligonucleotide  Insert the oligo into the chain using DNA ligase Protein Expression  use synthetic oligos to change on codon in the original gene  ligate into plasmid  transform into E coli  grow bacteria, lyse cells harvest protein (your protein is expressed with all other proteins)  need a high level of expression for research samples Protein purification  your desired protein is mixed with a lot of other proteins and can contain fusion “carrier” proteins, His tags and WB-Tags  fusion or carrier proteins (therodoxin) are often contained in expression vectors which make your protein water soluable  one step enzymatic cleave (e.g thrombin) is used at final steps to cleave off fusion protein (the one step will cleave the fusion protein + one his tag  His tags (sequence of 6-10 his residues) may be included  Nickel affinity columns recognize his tags and bind this protein exclusively. After other proteins are eluted, imidazole is used to elute protein of interest  You should already know some characteristics in your protein to show that you have purified it properly  Should appear as an extra band at the expected molecular weight  Use “western blot with antibody to confirm identity Gel electrophoresis  Separate proteins on a gel matrix with an electric field  Proteins migrate at different rates because of molecular weight (smaller ones move further)  Protein mixture can be visualized as individual components  SDS- micelle forming lipid detergent and solubilizes proteins. Has a 12 carbon chain with a Sulfate at the end. Sulfate makes it polar and soluble in water. SDS will make the protein linear and thus the protein will not have any function.  Polyacrylamide: polymeric gel matrix to embed proteins (the gel s what the proteins shift through)  Blue stain will stain all proteins. Western blot will only stain your protein. Diagram*** Protein engineering of subtilisin  Subtilisin is a endopeptidase cleaves peptide chains. Uses a serine to do it.  Secreted in large amounts from Bacillus species  Active site features a charged relay network involving an Asp His and Ser  The N on His is bonded to the Asp carboxylate side chain and the other N on His is bonded to the OH proton of Ser  Ser becomes activated because of the charge separation of OH and its O atom becomes more nucleophiic  Ser O atom attacks incoming substrate assisted by a neighboring carboxyamdie side chain of Asn  Substrate is a peptide bond  it is cleaved Mutagenesis of subtilisin  Method: choose a codon in the gene and perform randomized mutagenesis  use 25% of each nucleotide at each of three codon bases during oligonucleotide synthesis. By this method any three base codon can arise  wanted to engineering oxidative resistance. Subtilisin is easily oxidized but is stable to denaturants  the oxidation was happening at Met222 (next to active site Ser) results in 90% enzyme activity  methionine oxidized to methionine sulfoxide  Met 222 is a conserved residue in all subtilisins which suggest a functional role. Also shown through the inactivity protein after oxidation  Randomized mutagenesis w
More Less

Related notes for BCH210H1

Log In


Don't have an account?

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.