Difference between revisions of "Part:BBa K1921005"

Revision as of 05:21, 20 October 2016

GCW21

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 301
Illegal AgeI site found at 571
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 62

Usage

As a cell wall protein of Pichia pastoris, GCW21 is often used as an anchor protein in Pichia pastoris surface display system because of its stable connection with the dextranomer in cell wall. By fusing GCW21 with the target protein expression, the target protein can be displayed on the outer of the yeast cell wall.
By expressing the fusion protein of GCW21 protein and PETase, PETase was expressed on the surface of Pichia pastoris, and the whole cell catalyst for the degradation of PET was obtained. We made the PETase fixed on the cell wall, which can improve the stability of PETase, and is easy to control the degradation reaction of PET and PETase recycling.

Biology

This part was gained from Pichia pastoris GS115.
As one of the Glycosylphosphatidylinositoled cell wall proteins (GPI-CWPs), GCW21 is located in the outer layer of yeast cell wall, its C terminal is oligo mannose glycosylated. Subsequently, the mannose chain of GCW21 connect with the β-1,6 dextranomer of inner cell wall layer by forming covalent connection, thus, the GCW21 is fixed in the outer layer of the cell wall protein.

Reference

[1] Kinoshita T, Fujiata M. Overview of GPI biosynthesis [J]. The enzymes. 2009;26:1-30.
[2] Orlean P, Mennon AK. Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids [J]. Journal of lipid research.2007;48(5):993-1011.
[3] Mouyna I, Fontaine T, Vai M, et al. Glycosylphosphatidy linositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall[J]. Journal of Biological Chemistry.2000;275(20):14882-14889.

HPLC Results

Figure 1. The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration. Figure 1.The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.

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-'''Figure 1.'' The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
+'''Figure 1.''' The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.
 '''Figure 1.'''The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG. <br>