A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

Abstract

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexahistidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Functional and immunological evaluation of two novel proteins of Leptospira spp.

“…The acquisition of hostderived PLA by leptospiral receptor-bound PLG can degrade fibronectin and laminin that may aid bacterial propagation (Vieira et al, 2009;. By data mining the available genome sequences of L. interrogans (Nascimento et al, 2004), we have identified novel hypothetical proteins, several of them described as PLGbinding proteins Oliveira et al, 2011;Souza et al, 2012;Vieira et al, 2010b). Lsa44 and Lsa45 proteins are also able to bind PLG that is converted to functional PLA in the presence of uPA.…”

“…The gene sequence was amplified without the signal peptide tag. The PCR fragments of 1212 bp (LIC10645) and 1164 bp (LIC10731) were ligated into the E. coli expression vector pAE (Ramos et al, 2004) at the restriction sites presented in Table 1. This expression vector allows the inclusion of a His 6 epitope at the N-terminus of the recombinant proteins.…”

“…After adherence, pathogens have to overcome host tissue barriers to reach the blood circulation and target organs. Our group has reported previously that leptospires bind plasminogen (PLG) at their surface and that proteolytic activity is achieved due to the generation of plasmin (PLA), capable of degrading laminin and fibronectin (Vieira et al, 2009;.In the present study, we describe the cloning, expression, purification, and immunological and functional characterization of two novel predicted outer-membrane lipoproteins, encoded by the genes LIC10645 and LIC10731, identified in the genome sequences of Leptospira interrogans serovar Copenhageni by bioinformatics tools (Nascimento et al, 2004). The recombinant proteins were capable of adhering to laminin, and named Lsa44 (LIC10645) and Lsa45 (LIC10731) for leptospiral surface adhesin of 44 and 45 kDa, respectively.…”

“…In the present study, we describe the cloning, expression, purification, and immunological and functional characterization of two novel predicted outer-membrane lipoproteins, encoded by the genes LIC10645 and LIC10731, identified in the genome sequences of Leptospira interrogans serovar Copenhageni by bioinformatics tools (Nascimento et al, 2004). The recombinant proteins were capable of adhering to laminin, and named Lsa44 (LIC10645) and Lsa45 (LIC10731) for leptospiral surface adhesin of 44 and 45 kDa, respectively.…”

The Pyrococcus Exosome Complex

“…aRrp42 (PH1548) was amplified using primers aRrp42for (5Ј-AGGGATCCCATATGAGTGATA-ATGAGATCG-3Ј) and aRrp42rev (5Ј-ATCTGCAGATTAGGTA-AATATTACTCC-3Ј); the restriction sites for BamHI, NdeI, and PstI are underlined. NdeI-and PstI-digested DNA of aRrp42 was inserted into the expression vector pAE (15). aRrp4-aRrp41 were amplified using primer aRrp4for (5Ј-CGGATCCCATATGAAGAGGATTTTTGTTC-3Ј) and reverse primer for aRrp41 (aRrp41rev).…”