INP with 6His tag:

Part name: BBa_K2509019.

Short description: INP with 6His tag

Schematic diagrams (design):

Long description: This is an improved version of BBa_K584021 composed of 6His tag and partial sequence of INP. The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in Pseudomonas syringae and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. We replaced the coding sequence from 222aa to 1061aa of INP with a 6His tag flanked by BamHI and HindIII sites to make this part. Further we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET expression vectors. This part can be used in the surface display of recombinant proteins or small tags on Escherichia coli by replacing 6His tag with a target protein with or without a stop codon.

Source of the part: The partial coding region of INP is from BBa_K584021, and the 6His tag is a commonly used small amino acid motiff that consists of six histidine residues. The whole parts is synthesized by GenScript Corporation.

Design considerations: First we added an NdeI site before the start codon and an XhoI site after the final stop codon in order that it could be easily ligated into pET32 vector, secondary we added BamHI and HindIII sites at two sides of the 6His tag so that this small tag could be easily replaced by other proteins, and also we added a stop codon at the end of the 6His tag so that this part can only actually express an N part of INP fused with 6His tag at the C-terminus.

Results

Dot-Blot result and Western-blot result

Conclusion: This part was expressed correctly in E.coli.

Immunofluorescence (IF) results

Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed.

Binding results of E.coli strains and NTA beads

Conclusion: The 6His tag was expressed at the surface of E.coli strain as designed, and the modified host can bind with NTA beads.

References:

1. Fan LH, Liu N, Yu MR, Yang ST, Chen HL. Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration. Biotechnol Bioeng. 2011 Dec; 108(12):2853-64.

2. Eui-Joong Kim and Seung-Ku Yoo. Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein. Biotechnology Techniques. 1998 Mar; 12(3):197–201.

3. Kim EJ, Yoo SK.Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein. Lett Appl Microbiol. 1999 Nov; 29(5):292-7.

4. Chungjatupornchai W, Fa-aroonsawat S. Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein. J Microbiol. 2009 Apr; 47(2):187-92.

6His-ProteinA-out

Part name: BBa_K2509037.

Short description: 6His-ProteinA-out

Schematic diagram:

Long description: This part encodes an immunoglobulin G-binding protein A of Staphylococcus aureus with a 6His tag at its N-terminus. Protein A is a 42 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has found use in biochemical research because of its ability to bind immunoglobulins. This part is designed to express at the surface of E.coli by directly being ligated into pET32a Vector or fused with INP or other outer membrane proteins. These E.coli strains that expressed protein A at their surface are thought to have the ability of binding IgG expressing cells or B cells.

Source of the part: The main coding region except for the 6His tag of this part is from genomic DNA of Staphylococcus aureus (strain NCTC 8325).

Design considerations: A 6His tag is fused at the N terminus of this part for easier detection and purification. And two pairs of restriction sites (BamHI+HindIII and NdeI+XhoI) were added on either side of this part so that this part can be ligated with INP or directly ligated into pET32a vector.

INP-6His-ProteinA-out

Part name: BBa_K2509038

Short description: INP-6His-ProteinA-out

Schematic diagram:

Long description: This part encode a fusion protein composed of INP and protein A with 6His tag which is designed to be expressed at the surface of E.coli. We replaced the 6His tag of INP-6His (BBa_K2509019) with an IgG binding protein 6His-protein A (BBa_K2509037) to build this part. After being transferred with this part, the host E.coli strains is considered to have the ability of binding with IgG secreting cells.

Source of the part: BBa_K2509019 and BBa_K2509037

Design considerations: The whole fragment of BBa_K2509037 digested with BamHI and HindIII was directly ligated with INP without stop codon to make a ‘sandwich fusion’.