Novel Expression Vectors Based on the pIGDM1 Plasmid

Abstract

Escherichia coli is one of the most widely used hosts for the production of heterologous proteins. Within this host, the choice of cloning vector constitutes a key factor for a satisfactory amplified expression of a target gene. We aimed to develop novel, unpatented expression vectors that enable the stable maintenance and efficient overproduction of proteins in E. coli. A series of expression vectors based on the ColE1-like pIGDM1 plasmid were constructed. The vectors named pIGDMCT7RS, pIG-DM4RS and pIGDMKAN carry various antibiotic resistance genes: chloramphenicol, ampicillin or kanamycin, respectively. Two derivatives contain the inducible T7 promoter while the third one bears the constitutive pms promoter from a clinical strain of Klebsiella pneumoniae. The pIGDM1-derivatives are compatible with other ColE1-like plasmids commonly used in molecular cloning. The pIGDMCT7RS and pIGDM4RS vectors contain genes encoding AGA and AGG tRNAs, which supplement the shortage of these tRNAs, increasing the efficiency of synthesis of heterologous proteins. In conclusion, pIG-DMCT7RS, pIGDM4RS and pIGDMKAN vectors, with significantly improved features, including compatibility with vast majority of other plasmids, were designed and constructed. They enable a high-level expression of a desired recombinant gene and therefore constitute a potential, valuable tool for pharmaceutical companies and research laboratories for their own research or for the production of recombinant biopharmaceuticals. Keywords Expression vectors • Recombinant protein production • Bacterial expression system • E. coli Abbreviations bla gene AMPICILLIN RESISTANCE GENE cat gene Chloramphenicol resistance gene kan gene Kanamycin resistance gene MCS Multiple cloning site SD Shine Dalgarno sequence IPTG Isopropyl ß-D-1-thiogalactopyranoside CPH The yeast cyclophilin gene IFNA13 The interferon alpha 13 gene Ubi HGH: ubiquitin-human growth hormone fusion gene

A Type I Pullulanase from Geobacillus subterraneus: Functional Expression in Escherichia coli, Enzyme Characterization, Truncation, and Application

“…For some proteins, the differences in the vectors also affect the soluble expression of the protein. [ 28 ] Expression vectors pET‐28a and pGEX‐4T‐1 were employed to express Pul13552 in this study. The nucleic acid gel electrophoresis was employed to verify the gene of pul13552 ( Figure d).…”

“…[ 33 ] Besides, enzyme activity was greatly inhibited by α‐cyclodextrin (0.1%) and urea (2 M), but activated by ethylene diamine tetraacetic acid (EDTA) (5 mM) and dithiothreitol (DTT) (2 mM). For the activation by the EDTA, it has been reported that when the concentration of Ca 2+ and Mg 2+ increases, the activity of the Pul GT decreases, [ 28 ] wherefore, the existence of the EDTA could reduce the concentration of the metal ions to increase the activity.…”

“…Taken all, our results revealed that Pul13552 can hydrolyze pullulan, amylopectin, and soluble starch, which contains α‐1,6‐glycosidic linkages, but it had poor hydrolysis ability toward α‐1,4‐glycosidic linkages, suggesting that Pul13552 is the type I pullulanase. [ 28 ]…”

“…Taken all, our results revealed that Pul13552 can hydrolyze pullulan, amylopectin, and soluble starch, which contains 𝛼-1,6-glycosidic linkages, but it had poor hydrolysis ability toward 𝛼-1,4-glycosidic linkages, suggesting that Pul13552 is the type I pullulanase. [28] TLC and HPLC were employed to detect the enzymatic hydrolysates, which were applied to evaluate the enzyme hydrolysis characteristics (Figure 4b,c). Results exhibited that the hydrolyzed product from pullulan was maltotriose, not panose or isopanose.…”

Effect of membrane fusion protein AdeT1 on the antimicrobial resistance of Escherichia coli

“…Constitutively active promoters have been successfully, albeit infrequently, employed in E. coli for producing recombinant proteins [58][59][60][61][62][63] . We demonstrated that two constitutive promoters allowed constant production of sfGFP at low levels (i.e., no obvious green fluorescence in cultures by visual observation, in contrast to sfGFP under T7 promoter).…”