Thesis

Optimisation of Crystal Formation for the recombinant Fungal Immunomodulatory Protein (FIP-Lrh) from Tiger Milk Mushroom

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Background: FIP-Lrh is a fungal immunomodulatory protein from Tiger Milk Mushroom with
immunomodulatory and cytotoxic properties. Its biological properties can be further elucidated with
the availability of a crystal structure. However, crystal structures from only four FIPs have been
reported so far. As such, previous study at UCSI and MGVI on rFIP-Lrh crystallisation under two
conditions gave hexagonal, irregular, and microcrystal structures, which warranted further
optimisation.

Objectives: To produce recombinant rFIP-Lrh in E. coli BL21 expression system and to optimise
conditions for crystallisation of the rFIP-Lrh.

Methodology: E. coli FIP-Lrh/pET-28a(+) transformants cultured in LB broth were induced for
expression with IPTG. Total cell lysates containing soluble rFIP-Lrh were purified using Ni-NTA affinity
chromatography, dialysed, and subjected to further purification using size exclusion chromatography.
Protein crystallisation was performed using three screening kits: Crystal Screen
TM
1 and 2, PEG/ION
Screen
TM
1 and 2, and NeXtal Tubes Classics Suite. Further optimisation was performed to obtain a
refined crystal structure of rFIP-Lrh.

Results: Soluble rFIP-Lrh (14.9 kDa) was successfully produced with a yield of 100 mg/L of expression
culture. After a few rounds of screenings, two optimised crystallisation conditions successfully
generated orthorhombic crystals: (i) 0.2M potassium sodium tartrate tetrahydrate, 0.1M sodium
citrate tribasic dihydrate pH 5.6, 2.0M ammonium sulphate and (ii) 0.2M K/Na tartrate, 0.1M trisodium
citrate
pH
5.6,
2.0M
ammonium
sulphate.



Conclusion:

The project's objectives were achieved. To protect the crystals during storage and
subsequent X-ray diffraction analysis, suitable cryoprotectants (such as glycerol and polyethylene
glycol) will be used in the future. The X-ray diffraction data obtained can be used to generate the
biological 3D structure of FIP-Lrh for further functional analysis.

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Availability

Detail Information

Series Title

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Call Number

BM 23-011

Publisher

i3L, Jakarta : i3L, Jakarta., 2023

Collation

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Language

English

ISBN/ISSN

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Classification

NONE

Content Type

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Media Type

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Carrier Type

-

Edition

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Subject(s)

Tiger Milk Mushroom
Fungal Immunomodulatory Protein
FIP-Lrh
Protein Crystallisation

Specific Detail Info

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Statement of Responsibility

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Other version/related

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