Part:BBa_K4348002

AKR1C4_his

AKR1C4 (Aldo-Keto Reductase Family 1 Member C4) is a 5 beta oxoreductase that plays a role in the bile acid synthesis pathway in humans. We have attached a his tag to the protein for easy protein extraction and purification.

Introduction

The McGill iGEM team set out to develop a cholesterol lowering probiotic as a preventative for cardiovascular disease. Both endogenously synthesized cholesterol and dietary cholesterol end up in the gut, where they are absorbed and sent around the body. McGill iGEM’s project consists of developing a novel metabolic pathway to convert cholesterol, which is absorbed in the gut, into coprostanol, a molecule that cannot be absorbed and is thus excreted from the gut. The metabolic pathway consists of a three step pathway with four metabolites: cholesterol, which is converted to cholestenone, then coprostanone and finally coprostanol. We repurposed existing enzymes to engineer a metabolic pathway to do this conversion, then packaged it in a probiotic bacterium. By converting intestinal cholesterol into coprostanol, this probiotic bacterium can prevent cholesterol absorption as a preventative for high cholesterol-induced cardiovascular disease.

Results

We began by assembling our reactions following our tested reaction mixture to confirm the activity of our proteins. We started off by testing our proteins individually to see if they would be able to perform the hypothesized substrate to product conversion. To achieve this we incubated AKR1C4 with Alpha-Coprostanone for 16 hours and then performed an ethyl acetate extraction, derivatization, and resuspension before measuring enzyme activity on GC-MS.

Figure 1. GC-MS chromatogram of an in vitro assay containing AKR1C4 mixed with coprostanone. The GC-MS chromatogram shows the 0.01% coprostanol standard, which is the hypothesized product of AKR1C4’s conversion from coprostanone, along with the product of a reaction containing AKR1C4, 100µM coprostanone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.5), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37˚C. Without the need for an ion search, there is a peak on the chromatogram of the AKR1C4 in vitro assay directly underneath the coprostanol standard. This is evidence that AKR1C4 efficiently catalyzes the conversion from coprostanone to coprostanol.

We then ran a pop assay on AKR1C4, which is a protocol we developed to test proteins without protein purification, which builds off of a pre-existing protocol for protein expression. Liquid cultures of AKR1C4 protein-expressing E. coli are pelleted, washed with PBS, frozen at -80°C, resuspended in 2mL PBS. These samples are sonicated on ice, 3 times for 30 second bursts at an amplitude of 20kHz, then spun at 12000 rpm and 4°C for 20 minutes to release proteins into the supernatant. The protein-containing supernatant (potassium phosphate buffer) is extracted and has other reaction components added to it before being run on the GCMS.

This allowed us to test AKR1C4 and confirm whether it still maintains activity following our incubation of extract with the appropriate sterol, and cofactor.

Figure 2. GC-MS chromatogram of two AKR1C4 pop assays in E. coli mixed with coprostanone substrate. A GC-MS chromatogram showing the 0.01% coprostanol standard along with the product of two AKR1C4 reactions containing: 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer, 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37°C. AKR1C4 was added to the reaction using the pop assay protocol. The AKR1C4 reaction chromatogram shows a peak directly underneath the coprostanol standard, indicating product formation using the pop assay protocol.

Instead of incubating our reactions for 16h, we incubated the reactions for 1h to see if they would still have catalytic activity that would be detectable on the GC-MS.

This allowed us to test AKR1C4 and confirm whether it still maintains activity following our reduced incubation time.

Figure 3. GC-MS chromatogram of two in vitro assays containing AKR1C4 mixed with coprostanone incubated for 1 hour. A GC-MS chromatogram showing the 0.01% coprostanol standard, along with the product of three AKR1C4 in vitro reaction containing AKR1C4, 100µM coprostanone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.5), 0.2% Triton X-100, and 5% ethanol, incubated for 1 hour at 37°C. On all three AKR1C4 in vitro reaction product chromatograms, there is a peak directly underneath the coprostanol standard. This is evidence that even for reduced incubation times, AKR1C4 still catalyzes significant and detectable amounts of cholestenone into coprostanone.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 315
  Illegal NgoMIV site found at 780
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 403