Part:BBa_K4156126

HlpA with INP tag

HlpA is a binding protein from Streptococcus gallolyticus that binds to acetyl heparan sulfate glycoprotein (HSPG) on the tumor surface (specifically to syndecan 1, which is upregulated during carcinogenesis). HlpA has been shown to enhance the ability of engineered bacteria to penetrate colorectal tumor cells.

Characterization

HlpA is a new basic part of LZU-CHINA 2022, which we designed to facilitate better adhesion of engineered strains to cancer cells. In order to verify the function of this component, we characterized it. To demonstrate that HlpA enables better bacterial adhesion to cells, we constructed HlpA with mRFP to characterize HlpA. And characterization details can be found at BBa_K4156128

GEC-Guangzhou 2023

Description

We have decided to design/construct the INP-HIpA protein. Ice nucleation protein (INP) is a commonly used protein carrier in cell surface display technology. It has a large region located on the outside of the cell, which can be used for the display of other proteins. We will use this protein to display the HIpA protein, which is a histone-like protein derived from Streptococcus and can bind to heparan sulfate proteoglycans (HSPG) on the surface of tumors.

Usage and Biology

Figure 1 Design of the inp-hlpA.

We used INP as the basis for fusing the hlpA gene to get the INP-HlpA protein. We used promoter pT7 and terminator B0015 as a gene circuit control system to express INP-HlpA and transform the recombinant plasmid based on pET23b into E. coli Rosetta.

Characterization

Figure 2 Gel electrophoresis of the inp-hlpA.

Figure 3 Illustration of reaction principle.

Figure 4 The experimental results related to inp-hlpA.

Under the same conditions, we set up assessments for E.coli Rosetta and engineered bacteria E.coli Rosetta/pT7-HlpA. We put the bacteria together with two types of colorectal cancer cells (RKO and CT26) to observe the number of bacteria adhere the cancer cells.Quantitatively, our results showed that E.coli Rosetta without pT7-HlpA have about 42*10^5 CFU counts well of RKO cells and about 39*10^5 CFU counts well of CT26. In comparison, E.coli Rosetta with pT7-HlpA have about 82*10^5 CFU counts well of RKO cells lysate and about 82*10^5 CFU counts well of CT26 cells lysate. By calculation, fusion protein pT7-HlpA enhance the ability of E.coli to adhere to RKO cells by 195% and CT26 by 210%.

Potential application directions

This experiment proves that INP-HlpA can increase the adhesion of E coli to colorectal cancer cells. In the future, it can be applied to treat colorectal cancer, improving the treatment effect, reducing the side effects of 5-FU on other parts of the human body. It could to bring more effective treatment to patients with promising prospects for development.

References

Boleij, Annemarie, et al. "Surface-exposed histone-like protein a modulates adherence of Streptococcus gallolyticus to colon adenocarcinoma cells." Infection and immunity 77.12 (2009): 5519-5527. Dou, Jian-lin, et al. "Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein." Virologica Sinica 26 (2011): 409-417.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 330
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 154
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 733
  
• 1000
  COMPATIBLE WITH RFC[1000]