Part:BBa_K4348000

AKR1D1_his

A human 5-beta reductase used in bile acid synthesis. Used in the 2022 McGill project to catalyze the second step of the cholesterol -> coprostanol pathway, which is 4-cholesten-3-one to coprostanone. Coprostanol cannot be absorbed by the gut, which is a unique property.

Introduction

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Results

Figure 1. 12% SDS-Page gel of AKR1D1 IPTG induction optimization. SDS-Page was performed on AKR1D1 at various IPTG incubation times and temperatures. The expected length is 37kDa.

Figure 2. GC-MS chromatogram for the optimized derivatization of AKR1D1. The GC-MS chromatogram shows the 0.01% coprostanone standard, which is the hypothesized product of AKR1D1’s conversion from cholestenone, along with the product of a reaction containing AKR1D1, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37 ̊C.

Figure 3. GC-MS chromatogram of three AKR1D1 pop assays in E. coli mixed with cholestenone substrate. The GC-MS chromatogram shows the 0.01% coprostanone standard along with the product of a three AKR1D1 reactions containing, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 16 hours at 37°C. AKR1D1 was added to the reaction using the pop assay protocol. The AKR1D1 reaction chromatogram shows a peak directly underneath the coprostanone standard, indicating product formation using the pop assay protocol.

Figure 4. GC-MS chromatogram of two in vitro assays containing AKR1D1 mixed with cholestenone incubated for 1 hour. A GC-MS chromatogram showing the 0.01% coprostanone standard, along with the product of two AKR1D1 in vitro reaction containing AKR1D1, 100µM cholestenone, 500µM NADPH, 100mM potassium phosphate buffer (pH 6.0), 0.2% Triton X-100, and 5% ethanol, incubated for 1 hour at 37°C, a significantly shorter incubation time than the previously-run 16 hours. The asterisk denotes that the concentration of protein, and substrate was doubled for this particular reaction. On both AKR1D1 in vitro RIT reaction product chromatograms, there is a peak directly underneath the coprostanone standard.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]