Advantages Of Dfr From Bl21 Ecoli Using Column Chromatography

The goal of this lab was to create and purify DHFR from BL21 ecoli using column chromatography. To know if we isolate DHFR their will be one band at 53 KD or above 50 KD and in our desalted eluate page should have the highestOD₂₈₀ which translates to the highest enzyme activity .Column chromatography is a method used to purify individual chemical compounds from mixtures of compounds. The main advantage of column chromatography is the relatively low-cost and disposability of the stationary phase used in the process.This process is used to manufacture prescription drugs such as for cancer
GST (glutathione s-transferase) which adds to increase solubility. DHFR (Dihydrofolate reductase), is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid,required for synthesis,nucleotide,amino acids, DHFR inhibition or reduction disrupts nucleic acids synthesis affecting cell growth, propagation using NADPH as electron donor, which can converted to the kinds of tetrahydrofolate cofactors. In humans, the DHFR enzyme is encoded by the
DHFR gene. DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in antibiotics.However, resistance has developed against some drugs, as a result of mutational changes in DHFR itself. SDS(sodium dodecyl sulfate) is a negatively charged detergent, breaks hydrogen bonds unfold proteins, disrupts secondary, tertiary structures which help with purify DHFR.. It's used for determining the protein size,identify protein,determine sample purity,identify existence of disulfide bonds and quantify amounts of protein. The chromatography we use is affinity chromatography which can use on protein with natural ligands it involves a covalent attachment of affinity tag to a protein. The tag provides rapid specific clean up. Affinity chromatography allows automation of protein purty.

In this lab to get our protein purifies we need to culture our colonies and induce to get an isolated one.Then we lyse the cells with a frozen protein and thaw enzymatically.
Next we centrifuge to separate soluble from insoluble then we do affinity chromatography which the nickel sticks to the His-tag, after that eluate imidazole, nxt we do size exclusion which separates small from big,big comes off first the imidazole captures it in the column next we do spectrophotometry. Then SDS-PAGE electrophoresis, size purity,enzyme test does its function. Finally we did DHFR enzymatic assay. In the SDS-PAGE electrophoresis we load the gel up with Laemmli buffer, precision plus protein standard,desalted eluate page, eluate page, wash page, flowthrough page, soluble page, insoluble page, inducted page, and uninducted page. The desalted eluate page is the sample of interest since it should have the highest OD₂₈₀ and have the highest enzyme
activity.
When we ran our gel there was nothing in that lane for desalted eluate page then the same for precision plus protein standard, and eluate page which is not good does not mean there was no enzyme activity their. We did the DHFR enzymatic assay to see if there was any changed when we did the assay for three minutes and 20 seconds or two hundred and ten seconds recording the number every 30 seconds we did the control and soluble page. The control start at .224 and the end of 210 second mark we got .221 the soluble page started at .436 and end of the 210 second mark we got .428. We decide that we had enzymatic activity but not where we wanted to get it at.