Part:BBa_K3388015

6xHis

The 6xHis tag, also known as polyhistidine tag, His6 tag and/or hexa histidine tag, is an amino acid motif consisting of at least 6 histidine residues fused to the carboxyl (C-) or amino (N-) terminus of a target protein in transfected cells.

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]

The Characteristic of UGT89C1 Protein：
Molecular weight（kD）：50.49
Isoelectric point：5.67
Large Average Hydrophilicity：-0.039
Instability index：39.84
Half life period：>10h
1.plasmid construction
Due to the influence of the COVID-19 epidemic, we have little time to do the cloning. Therefore, we sent our plasmid file to Genescript, which helped us optimize the gene sequence and cloned it into the target vector. We asked Genescript to clone UGT78D2 gene into pGEX-5X-3, and send us freeze-dried plasmid and glycerol bacteria.
2. Gel imaging
According to the instructions, we performed gel imaging on Escherichia coli DE3 transformed with pGEX-UGT78D2 plasmid and pCDF-UGT89C1 plasmid. As shown in the figure, the band of the sample was compared with marker, which proved that we successfully introduced the target gene into bacteria.
<img src="https://parts.igem.org/File:T--NWU-CHINA-B--ningjiao.jpg" />
3.Glycosylation module experiment
3.1Strains and growth
Using E. coli DE3 as the expression host, 2ml seed solution was prepared. Take the 100-fold diluted seed solution, transfer it to liquid LB medium, and grow at 37°C so that its OD value reaches 0.8.
3.2Optimize induction conditions
Take each 10ul seed solution in 5 bottles of 10ml liquid LB medium (containing Am). It was grown at 37°C and 220 rpm to an OD600 of 0.8. Add 10ul each of IPTG at different concentrations (0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM) to 5 bottles of bacterial solution, and label them.
Induce protein expression at 18°C. Take 100ul every hour to measure and record the OD of the bacterial solution at each IPTG concentration for a total of 10 hours. Take another 0.5ml of bacteria liquid every hour and add 4.5ml of sterile water, and mix it as the dilution of 10^-1; and so on, get 10^-2, 10^-3, 10^-4, 10^-5, 10^-6 dilution of the bacterial solution, from the last three dilutions of the bacterial solution, take 200ul of Am antibiotic-coated plates, incubate overnight at 37°C, count the plates to obtain the number of bacteria per hour at each inducer concentration, and record. After 20h, purify the protein at each IPTG concentration (GST-tagged protein affinity chromatography kit and 6xHis-tagged protein kit, small amount extraction), measure the concentration with nanodrop, and record.
Due to the poor expression effect of inducing protein UGT78D2 for the first time, we consulted XJTU-China team, and they helped us improve the experimental scheme as follows: Add 10ul IPTG with concentrations of 0.2mM, 0.4Mm, 0.6mM, 0.8mM and 1.0mM to 10ml liquid LB medium in a 150ml conical flask. Growing at 18℃ for 20 hours, measuring and recording the OD of bacterial liquid at different IPTG concentrations every 1 hour from the first hour, for a total of 20 hours. After 20 hours, the proteins with different IPTG concentrations were purified (GST tag protein affinity chromatography kit, a small amount of extraction), and the concentrations were measured by biodrop and recorded.
3.3 Induction of protein expression
Induce a large amount of protein expression with the optimal inducer concentration. After 20 hours, purify the protein at each IPTG concentration (GST-tagged protein affinity chromatography kit and 6xHis-tagged protein kit, mass extraction), measure the concentration with nanodrop, and record.
3.4 Electrophoresis of protein
We electrophorese the UGT78D2 protein after affinity chromatography together with the detection solution retained during chromatography. As shown in the figure, we compared the bands of protein marker and protein eluent lane, which proved that we got the target protein UGT78D2.
<img src="https://parts.igem.org/File:T--NWU-CHINA-B--dianyong.jpg" />
3.5 Protein ultrafiltration
We ultrafiltered protein by Amicon Ultra-15 10K device according to the instructions. The protein was divided into 5 equal parts, and each part was concentrated with Tris-HCl buffer with pH 6.0, 6.5, 7.0, 7.5 and 8.0 respectively.