The method of obtaining the expression cassette vector, expression vector and expression system for the expression and secretion of heterologous proteins constitutive production

摘要

The method of obtaining the expression cassette vector, expression vector and expression system for the expression and secretion of heterologous proteins constitutive production.The present invention is a method of obtaining the vector expression pt7bsxa through the stages (a) the inclusion of sites of specific restriction; (b) construction, directed evolution, the library of variants with mutations in the region of the promoter for random error prone p Cr; (c) construction, directed evolution,The library of variants with random mutations in the region of origin of replication (ORI) co1e1 by error prone PCR and (d) insertion of the gene of the protein of interest, in particular, enzymes oxidorredutases, transferases, hidrolases, liases, isomerases and / or ligases. In addition, the present application relates to a tape of expression composed of (a) variants of the promoter region bsxa (SEQ ID No.12a, SEQ ID No. 27a, SEQ ID No. 30a, SEQ ID No. 32a, SEQ ID No. 33A, SEQ ID No. 34a, SEQ ID No. 38a, SEQ ID No. 39A and / or their combinations obtained by site directed mutagenesis, such as SEQ ID No. 40A (RPA), SEQ ID No. 41a (RPB), SEQ ID No. 42 (rpab)), (b) variants of the source region R Eplicau00e7u00e3o (ORI co1e1) (SEQ ID No. 50, SEQ ID No. 60, SEQ ID No. 70, SEQ ID No.80) and (c) a gene of a protein of interest, a vector of expression pt7bsxa, obtained by said production method, containing the tape of expression and an expression system for the constitutive production and secretion of heterologous proteins, in particular, enzymes oxidorredutases, Transferases, hidrolases, liases, isomerases and / or ligases, preferably.The gene of the enzyme xylanase wild - type (xy1a) in Escherichia coli.