Part:BBa_K5366030

T7 promoter-RBS-TrxA-linker-AJC7-6xHis -T7 termonator

TrxA pro-fusion labeling and AJC7 structural gene co-expression plasmid

Construction

1.The pro-fusion tagged TrxA was PCR amplified and the pET-28a(+)-AJC7 vector was linearised by reverse PCR, running nucleic acid gels of the correct fragments for gel recovery. The plasmid structure is shown in Figure1.

Fig.1 Map of pET-28a(+)-TrxA-AJC7 plasmid

2.The pro-fusion tagged fragments were recombined with the vector through homologous recombination, followed by heat-stimulated transformation. Individual transformed colonies that grew on the plates were then selected and cultured. Colony PCR was conducted on these colonies to confirm the presence of the recombinant plasmid, as illustrated in Figure 2.

Fig.2 Nucleic acid gel map of colony PCR

Indicator

1.Single colonies confirmed to have successful colony PCR were subsequently sent for sequencing. Those colonies that exhibited the correct sequencing results were subjected to induced expression. Following the induction, both the supernatant and precipitates obtained after cell lysis were analyzed using SDS-polyacrylamide gel electrophoresis, as shown in Fig. 3. The molecular weight of TrxA-AJC7 was determined to be 70.4 kDa. Notably, with the addition of the fusion-tagged MBP (lane 3), there was a significant increase in the concentration of the 70.4kDa protein band in the supernatant of the cell lysis solution (lane 4), while the corresponding precipitated protein band appeared much lighter.

Fig.3 SDS-PAGE of recombinant AJC7 with a fusion-promoting tag(M: protein marker; Lane 1: precipitation of AJC7; Lane 2: supernatant of AJC7; Lane 3: precipitation of TrxA-AJC7; Lane 4: supernatant of TrxA-AJC7)

2.induced at 20℃ for 16h, the protein gel was further analysed by optical density analysis to quantify the increase in protein expression, and the results were analysed in Figures 4 and 5 below:

Fig.4 Histogram of densitometry data of SDS-PAGE gel supernatant and precipitated protein bands of bacterial cell breakage fluid before and after fusion-promoting tag attachment

Fig.5 Percentage comparison of densitometry data for bacterial cell breakage, supernatant, and precipitated protein bands before and after fusion-promoting tag attachment

Result

The data presented above demonstrate that under specific induction conditions, the pro-fusion tag TrxA significantly enhanced the solubility of the proteins, achieving a 6.79-fold increase compared to AJC7. Furthermore, the density values of the protein bands in the precipitates were markedly reduced. This indicates that the fusion-promoting tag TrxA is more effective in enhancing the solubility of protein AJC7.
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1896
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 902
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 363
  Illegal AgeI site found at 1404
  
• 1000
  COMPATIBLE WITH RFC[1000]