TROUBLESHOOTING THE HETEROLOGOUS EXPRESSION OF RIBOFLAVIN SYNTHASE FROM PHOTOBACTERIUM SP. J15
Gol Mohammad Dorrazehi, Laila Noh, Mohd Shukuri Mohamad Ali, Raja Noor Zaliha Raja Abd Rahman, Abu Bakar Salleh, Normi Mohd Yahaya and Thean Chor Leow*
ABSTRACT
Riboflavin synthase is an enzyme involved in riboflavin biosynthesis pathway that catalyzes the formation of
riboflavin (vitamin B2). Riboflavin synthase is a homotrimeric structure that consists of three subunits. The
enzyme catalyzes the riboflavin production from two substrate of 6,7-dimethyl-8-ribityllumazine. Riboflavin
synthase of many microorganisms such as B. subtilis and E. coli has been studied previously and the structure and
mechanism of this enzyme was reported. However, till date there has been no report on riboflavin synthase from
marine bacteria. In this study, a 612 bp gene of riboflavin synthase was isolated from Photobacterium sp. J15. The
gene was amplified by PCR and cloned into pTrcHis and pET-32b(+) vectors, respectively. The expression of
riboflavin synthase gene in E. coli Top10 using pTrcHis system was very low even at insoluble fractions. Analysis
of the DNA sequence of this riboflavin synthase shows 6 rare codons, in which common E. coli strains are not
able to supply the corresponding tRNAs; thus the E. coli strain Rosetta-gami B (DE3) pLysS was selected as the
expression host, which is able to supply rare tRNAs by carrying a chloramphenicol resistant plasmid (pRARE2)
and also enhance disulfide bond formation in cytoplasm. The riboflavin synthase was expressed in the cytoplasm
of recombinant clone at high level in inclusion body fraction. The identified bands of riboflavin synthase in SDSPAGE
shows the size of ~40 kDa, which represent the riboflavin synthase (~23 kDa) tagged with Trx-Tag (~12
kDa), S-Tag (~4 kDa) and His-Tag (~1 kDa). The overexpression of proteins provides the sufficient amount of
enzyme for further analysis such as charachterization, structure studies through protein crystallography and finally
inhibition studies.
Keywords: Riboflavin synthase, heterologous expression, rare codons.
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