Yeast surface display technology has been used in many fields. Among them, enzyme display represents an effective whole-cell biocatalyst and shows good potential in industrial biotransformation. As an expert in the field of microbial surface display, Creative BioMart has developed a surface display technology based on the cell wall protein Sed1p of Pichia pastoris. Through this technology, we are able to achieve efficient enzyme display and improve the properties of the displayed enzyme. This technology may pave the way for the development of whole-cell biocatalyst based on the display system.
In order to display the target enzyme on the surface of Pichia pastoris, we fuse the sequence of Sed1p with the C-terminal sequence of GOI, and insert a His-tag sequence encoding six amino acid residues between the sequences of GOI and Sed1p. The above fragments are connected with the expression vector (we usually use the pPICZαA vector by default) and electroporation is inserted into Pichia pastoris for chromosome integration. Finally, the recombinant protein is expressed under the control of the pAOX1 promoter.
According to the enzyme activity and property test we conducted, the properties of the displaying enzyme (such as the optimal temperature and pH) are likely to be improved.
| Generation and amplification of expression vector | Timeline |
|
2-3 weeks |
| Linearization vector and electroporation into Pichia pastoris | |
|
1 week |
| Optimization of expression conditions | |
|
1 week |
| Amplification, expression and purification of target protein | |
|
1-2 weeks |
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