Purification of Bacterial 4-Hydroxyphenylpyruvate Dioxygenase | CHEM 603, Lab Reports of Biochemistry

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Experiment 2

PURIFICATION OF

BACTERIAL 4-HYDROXYPHENYLPYRUVATE

DIOXYGENASE

A 280nm NaCl (mM) Fraction 40 KDa

30 KDa

Experiment 2a INOCULATION OF LARGE-SCALE CULTURES In this short lab, each researcher will inoculate a fresh large-scale culture with the small- scale cultures grown in the previous lab. This large scale culture will then be used in the subsequent protein purification steps to isolate bacterial HPPD. Antibiotic resistance in bacteria is a serious problem facing society today. There are many reasons for this problem, one of which is an overuse of antibiotics. In hospitals where many people have compromised immune systems, resistant bacteria often kill a people with otherwise minor ailments. β-lactam antibiotics, such as the penicillins and the cephalosporins, are among the most commonly used antimicrobial agents. The production of β-lactamases, which catalyze β-lactam hydrolysis, is the predominant mechanism of bacterial resistance to these antibiotics. Here we select for those bacteria that can produce β-lactamase from a second gene in our recombinant plasmid and thus hydrolyse ampicillin (left) in the medium. The β-lactamase that is made is secreted into the periplasmic space and acts as a barrier against diffusing ampicillin (see Figure on the next page). In this way we allow only those cells that also contain the HPPD gene to grow since both genes are part of a single circular plasmid of DNA that we have constructed. Once the cells have reached a target density in the media, we will switch on the production of HPPD using a chemical known as iso-propyl-β-galactopyranoside (IPTG). The pET17b plasmid that we placed our HPPD gene in, is one of a series of plasmids that use the pET expression system. The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription. Expression is induced by providing a source of T7 RNA polymerase in the host cell. This is the function of IPTG, which induces the production of T7 polymerase from a man made “lac” based operon inserted into the host’s ( E. coli ) genome. T7 RNA polymerase is so selective and so highly active that almost all of the cell's resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. S CH 3 CH 3 O HO H N O NH 2 O

Today’s Strategy Inoculate large scale cultures Induce cells with IPTG and allow to express HPPD Harvest cells by centrifugation Prepare Buffers for Experiments 2b and 2c

INOCULATION

1. 2 x 100 μL from each of your small-scale cultures has been spread onto LB agar, 100 μg/mL ampicillin.
2. Resuspend cells from the plates in 20 mL of LB Broth and use to inoculate 1000 mLs of LB Broth, 100 μg/mL ampicillin (try to minimize the time taken to inoculate as longer times will tend to put the cells into some stasis stage that will cause your culture to lag)
3. Incubate this large-scale culture at 37 o C with shaking (225 rpm)
4. Monitor the growth of the cells by observing the absorbance (light scatter) at 600 nm. Dilute 0.25 mL of cells into 1.75 mL of water for each OD600nm measurement. You will use the Shimadzu spectrophotometer in photometric mode (see below) i.e., read the absorbance at just one wavelength, 600 nm. In photometric mode press F2 to define your measurement parameters. NB: Strickly speaking we are not measuring absorbance, we are instead measuring light scatter off of the cells, the instrument, of course can’t tell the difference. All it knows is that less light passes from the sample than what was incident upon it. Scatter has a quite low linear response, you will have an accurate measure of cell density provided the absorbance value is below 0.25 Abs units. To avoid excessive scatter, each time you make a measurement, dilute your sample by a factor of four in water. Spectrophotometer Tool Bar

Record and Plot your Cell Growth Time (min) OD (600nm) Time (min) OD (600nm) -0. 0

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-100 0 100 200 300 400 500 OD 600nm Time (min)

PREPARATION OF BUFFERS FOR EXPERIMENTS 2B AND 2C

Prepare 1.5 L of 50 mM HEPES buffer.

1. In a 2L beeker add HEPES powder sufficient to make a 50 mM solution in 1.5 L
2. Add ~1200 mL of water and stir until dissolved.
3. Standardize the pH electrode (see TA) and insert into HEPES solution while stirring.
4. Titrate strong acid or base to bring the pH to 7.0 at RT.
5. Bring the volume to 1500 mL.
6. Add EDTA to a final concentration of 1 mM.
7. Place 500 mL of the solution in a bottle and label descriptively GROUP 1(T or R) (Buffer A, 50 mM HEPES, 1mM EDTA, pH 7.0 (DATE)).
8. Place another 500 mL of the solution in a bottle and add NaCl to a final concentration of 500 mM. Label descriptively GROUP 1(T or R) (Buffer B, 50 mM HEPES, 500 mM NaCl, 1mM EDTA, pH 7.0 (DATE)).
9. Place the remaining 500 mL in a separate bottle and label descriptively GROUP X(T or R) (Resuspension Buffer, 50 mM HEPES, 1mM EDTA, pH 7.0 (DATE)) and store this bottle also.