Part:BBa_K5366029

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

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

Construction

1.The pro-fusion tagged MBP 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.

 Figure1  Map of pET-28a(+)-MBP-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 MBP-AJC7 was determined to be 99.0 kDa. Notably, with the addition of the fusion-tagged MBP (lane 7), there was a significant increase in the concentration of the 99.0-kDa protein band in the supernatant of the cell lysis solution (lane 8), 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 7: precipitation of MBP-AJC7; Lane 8: Supernatant of MBP-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 MBP significantly enhanced the solubility of the proteins, achieving a 6.92-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 MBP 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 2671
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 399
  Illegal BglII site found at 1677
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2179
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 97