ABSTRACT
Background: hepatitis C virus [HCV] is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma [HCC]. Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses
Objectives: in current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems
Materials and Methods: the expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer
Results: the data showed a clear band of [tilde]573 bp for HCV core and [tilde]2238 bp for core-E1E2 genes in agarose gel. Moreover, a [tilde]21 kDa band of core protein and a [tilde]83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method
Conclusions: this study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system
ABSTRACT
Tissue plasminogen activator [tPA] is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli [E. coli] is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm
Subject(s)
Escherichia coli , Periplasm , Cytoplasm , Polymerase Chain ReactionABSTRACT
The pLE2SCX vector was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. The pLE2SCX construct contains three independent selection markers: herpes simplex virus thymidine kinase [HSV-TK], cytosine deaminase [CD] and streptothericin acetyltransferase gene [sat] in multiple cloning site, flanking by 5' and 3' untranslated regions of the previously cloned Leishmania major hexa-binding protein gene. Selection was based on resistance to the nourseothericin [Ns] which corresponds to sat gene. The two negative selection; HSV-TK and CD genes, make the transformed cell sensitive to ganciclovir [GCV] and 5-fluorocytosine [5-FC]. The vector was introduced into Leishmania promastigotes by electroporation and maintained as circular form. The selected transfectants were not grown on media with GCV or 5-FC. Using two drug sensitive selectable markers together on a vector is a novel strategy in gene cloning in Leishmania. This stable transfection vector has allowed the permanently expression of several different exogenous genes at the same time in Leishmania