<span style="font-size:11.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US;mso-bidi-language: HI" lang="EN-GB">Rare codons caused poor expression of mammalian cell entry (Mce1A) gene cloned from <i>Mycobacterium leprae</i> in <i>Escherichia coli</i></span>

64-68<span style="font-size:11.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-gb;mso-bidi-language:="" hi"="" lang="EN-GB">Mammalian cell entry gene of mycobacterium helps its entry into epithelial and natural target cells. In order to carry out the functional assay of Mycobacterium leprae Mce1A, the whole gene was cloned in Escherichia coli. An overexpression vector carrying M. leprae mce1A gene, under IPTG inducible T5 promoter, was cloned in E. coli for expression of encoded protein with N-terminal 6xHis-tag. In the absence of IPTG, E. coli cells carrying the mce1A gene grew normally but induction of gene expression led to inhibition of cell growth. Western blot analysis using anti-His HRP conjugate showed full length Mce1A protein expressed in low amount. Deletion of N-terminal region having adjacent arginine rare codons resulted in overexpression of truncated protein as inclusion bodies without inhibiting cell division with size reduction of recombinant protein. However the full length protein poorly expressed without size reduction.</span

Cloning of mce1 locus of Mycobacterium leprae in Mycobacterium smegmatis mc2 155 SMR5 and evaluation of expression of mce1 genes in M. smegmatis and M. leprae

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of &#216;C31. Transformation of this plasmid into Mycobacterium smegmatis mc2 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5'TTG disrupting the gatA gene (Glu-tRNAGln amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient's biopsy showed that mce locus is transcribed as an operon in the pathogen also