RESUMO
Expressions of several genes in bacteria were carried out by independent promoter. However, in case of eukaryotes ribosome skipping and introduction of IRES are employed as alternative to multiple translation initiation. Foot and mouth disease virus (FMDV) 2A peptide has been widely used for co-expression of multiple genes in eukaryotic, plant and mammalian systems. The 18 amino acid 2A peptide of FMDV facilitates efficient co-translational dissociation of the polyprotein into discrete protein products. To study the role of 2A in multimeric protein production a construct consisting of tandem repeat of 4 units of C- terminal VP1 linked through 2A sequence was made and expressed in E. coli. Along with tetramer protein, trimer, dimer and monomer proteins were produced. Stability studies showed that the tetramer protein was cleaved to smaller monomer on storage. The results provide scope for using FMDV 2A for expressing multiple genes under a single promoter in prokaryotes.
Assuntos
Animais , Clonagem Molecular , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Febre Aftosa/metabolismo , Vírus da Febre Aftosa/metabolismo , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Técnicas Genéticas , Peptídeo Hidrolases/química , Peptídeos/química , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Virais/químicaRESUMO
Economizing the research protocols by using low cost technologies is the need of laboratories of developing world. Screening of recombinant E. coli colonies is the crucial step in gene cloning and expression studies. In the present study, the cost effectiveness of colony lysis method and colony PCR method in the screening of recombinant E. coli colonies was compared. The colony lysis method was 20 two times more cost effective and less time consuming and can be used to screen the recombinant E. coli colonies in large scale instead of colony PCR method.