REKAYASA PROTEIN SUPEROKSIDA DISMUTASE Staphylococcus equorum REKOMBINAN, OVERPRODUKSI, DAN KARAKTERISASINYA
Recombinant manganese superoxide dimutase Staphylococcus equorum (rMnSODSeq) exists as homodimer. Enzyme rMnSODSeq has moderate thermal stability with dimeric melting temperature (Tm) of 52°C. Three dimentional structure of rMnSODSeq shows that monomers interaction can be enhanced to improve dime...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/44269 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Recombinant manganese superoxide dimutase Staphylococcus equorum (rMnSODSeq) exists as
homodimer. Enzyme rMnSODSeq has moderate thermal stability with dimeric melting temperature
(Tm) of 52°C. Three dimentional structure of rMnSODSeq shows that monomers interaction can be
enhanced to improve dimer stability. This research aimed to increase the rMnSODSeq dimer
structural stability by amino acid substitution and to study the effect of the substitution to its
stability and activity. The research was initiated by in silico modelling using COOT program to
determine the substitution site and the modelling resulted Glycine-125 to Threonin (G125T).
Substitution of G125T was done at DNA level by site-directed mutagenesis of GG to AC of
rMnSODSeq coding region in pJexpress414_sod using Polymerase Chain Reaction with two Single
Primer IN Reaction Parallel. Mutation product was confirmed using sequencing and the desired
mutation was successfully obtained using annealing temperature at 59°C. Overexpression of
rMnSODSeq_G125T was performed at 37°C 150 rpm with IPTG induction at final concentration of
1 mM for 4 hours. Proteins were purified using affinity chromatography Ni2+ -NTA continued with
anion exchange chromatography Q-Sepharose yielded pure protein. The pure protein was then
characterized to determine its activity and dimer structural stability. Decreased activity of
rMnSODSeq_G125T was observed by zymographic and colorimetric assay with specific activity
reduction of 90.58% from 1730.3 U/mg (rMnSODSeq) into 162.96 U/mg (rMnSODSeq_G125T). The
thermal shift assay showed that both proteins had Tm value of 58°C hence no difference in dimer
structural stability. The G125T substitution of rMnSODSeq does not increase the dimer structural
stability and causes reduction in protein specific activity.
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