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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 10:28, 25 September 2024

_ pPICZαA-Profilin 3-sfGFP

pPICZαA-Profilin 3-sfGFP


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 193
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 427


Origin

Synthesized by company and constructed by the team.

Properties

Fusion expression of protein Profilin3-GFP.

Usage and Biology

The pPICZαA vectors are 3.6 kb plasmids designed for the expression and secretion of recombinant proteins in *Pichia pastoris* yeast. These recombinant proteins are expressed as N-terminal fusions with the coding sequence of the α-factor secretion signal derived from *Saccharomyces cerevisiae*. The vectors facilitate high-level gene expression induced by methanol in *Pichia pastoris* and can be utilized with any strain of this yeast. We inserted the Profilin 3 coding gene into this plasmid for fusion expression with GFP.

Cultivation, Purification and SDS-PAGE

plasmid diagram of pPICZαA-Profilin 3-sfGFP
Figure 1. Plasmid diagram of pPICZαA-Profilin 3-sfGFP.

The vector pPICZαA originates from the *Pichia pastoris* expression vector. It is methanol-inducible and allows high-level expression and secretion of recombinant proteins, specifically in yeast. This vector was chosen to measure the DNA expression of Profilin 3 (PFN3) in yeast.

PCR amplification of the fragment used for plasmid construction
Figure 2. PCR amplification of the fragment used for plasmid construction.

We constructed pPICZαA-Profilin 3-sfGFP using homologous recombination. The Profilin 3-sfGFP sequence was amplified by PCR with a length of 1146 bp.

Growth of plasmid pPICZaαA-PFN3 transformed bacteria on LB agar plates
Figure 3. Growth of plasmid pPICZaαA-PFN3 transformed bacteria on LB agar plates.

Secondly, the correct plasmids of pPICZαA-PFN3-sfGFP were transferred into yeast. It was cultured on YPD medium agar plates overnight. The image shows significant bacterial growth on YPD agar plates with easily observable individual colonies.

Growth of pPICZαA-PFN3-sfGFP in yeast on YPD agar plates
Figure 4. Growth of pPICZαA-PFN3-sfGFP in yeast on YPD agar plates.

Finally, the protein expression of pPICZαA-PFN3 was tested by SDS-PAGE. The results are shown below, displaying the target band for Profilin 3 (15 kDa).

SDS-PAGE analysis of PFN3 expression in yeast cells
Figure 5. SDS-PAGE analysis of PFN3 expression in yeast cells.

Measurement and Characterization

We used DNA sequencing to determine the full nucleotide sequence of our reconstructed plasmids, verifying the accuracy and integrity of the pPICZαA-PFN3-sfGFP plasmids.

Sanger sequencing result of pPICZαA-PFN3-sfGFP plasmid
Figure 6. Sanger sequencing result of pPICZαA-PFN3-sfGFP plasmid.

Reference

[1](https://www.thermofisher.cn/order/catalog/product/cn/zh/V19520)

[2](https://www.snapgene.com/plasmids/yeast_plasmids/pPICZ(alpha)_)