biotechnology-biomarkers-systems-biology-2019-tracks

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Page 28
Biochemistry & Molecular Biology Journal
ISSN: 2471-8084
Internat i ona l Conference on
Biotechnology, Biomarkers
& Systems Biology
M a r c h 0 4 - 0 5 , 2 0 1 9
Am s t e r d a m , N e t h e r l a n d s
Biotechnology, Biomarkers & Systems Biology 2019
UnaG protein from Japan eel (Anguilla japonica) is a novel fluorescent
protein with binding domain that acquires fluorescence when bound to
unconjugated bilirubin (UC-BR). In this study, several point mutations (F17M,
N57D, N57E, N57R, L41F, Y99F_Y134W, Y99M_Y134M, and W9F_W103F) were
made on the UnaG nucleotide sequence via using a method for sequence
and ligation independent cloning (SLIC). The aim of the mutations on UnaG
is to figure out the change in fluorescence properties. The new mutagenic
vector was transformed into the commercial competent cells (E. coli Mach1)
by using heat shock at 42 °C for 2 minutes. Transformed cells were grown
on and selected from the LB agar plate with ampicillin. (1:1000). The DNA
sequencing results show that all these mutations have done correctly. The
expression of the mutant proteins was made in the pTOLT expression system
by inducing with IPTG. Cells were collected with high speed centrifugation.
Before disrupting the cells, lysozyme enzyme was added to make break up
the cells easier, some protease inhibitors (phenylmethylsulfonyl fluoride,
benzamidine) were added for the protection from proteases of the protein
and DNase and RNase were added on the cell pellet to avoid the DNA and
RNA contaminations. Ultracentrifugation was applied on the cell lysate. Ni-
NTA affinity chromatography system was used to get the pure mutant proteins
from supernatant. SDS-PAGE and semi-dry Western blot were applied on the
protein for the qualitative analyse. The pure protein bands were observed on
the SDS-PAGE gel image. Additionally, the spectroscopic features of purified
mutant proteins were measured after adding fresh UC-BR on fluorescence
spectrophotometer. Excitation and emission spectra of the mutant proteins
are similar; even so they have different fluorescence intensity at the same
concentration. This study suggests that mutant UnaG proteins can be used to
detect UC-BR level of cells/tissue.
Biography
Numan Eczacioğlu has started his PhD at Karamanoğlu
Mehmetbey University, Turkey and still continues. Also, he is
working as a Research Assistant at Bioengineering Department
of the same university. He is the part of Tubitak and British
CouncilNewtonKatipCelebiFundbilateralcooperationprogram
with collaborate Newcastle University and Karamanoğlu
Mehmetbey University.
Generation and characterization of some UnaG mutant
Numan Eczacioğlu1, Yakup Ulusu1, Isa Gökçe2 and Jeremy H Lakey3
1Karamanoğlu Mehmetbey University, Turkey
2Gaziosmanpaşa University, Turkey
3Newcastle University, UK
Numan Eczacioğlu et al., Biochem Mol biol J 2019, Volume:5
DOI: 10.21767/2471-8084-C1-023