scholarly journals Use of Bacterial Artificial Chromosomes in Baculovirus Research and Recombinant Protein Expression: Current Trends and Future Perspectives

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Polly Roy ◽  
Rob Noad
Keyword(s):  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Short Review ◽  
Virus Genome ◽  
Bacterial Artificial Chromosomes ◽  
Baculovirus Genome ◽  
Artificial Chromosomes ◽  
Foreign Genes

The baculovirus expression system is one of the most successful and widely used eukaryotic protein expression methods. This short review will summarise the role of bacterial artificial chromosomes (BACS) as an enabling technology for the modification of the virus genome. For many years baculovirus genomes have been maintained in E. coli as bacterial artificial chromosomes, and foreign genes have been inserted using a transposition-based system. However, with recent advances in molecular biology techniques, particularly targeting reverse engineering of the baculovirus genome by recombineering, new frontiers in protein expression are being addressed. In particular, BACs have facilitated the propagation of disabled virus genomes that allow high throughput protein expression. Furthermore, improvement in the selection of recombinant viral genomes inserted into BACS has enabled the expression of multiprotein complexes by iterative recombineering of the baculovirus genome.

scholarly journals Construction of a Low-Temperature Protein Expression System Using a Cold-Adapted Bacterium, Shewanella sp. Strain Ac10, as the Host

2007 ◽  
Vol 73 (15) ◽  
pp. 4849-4856 ◽  
Author(s):  
Ryoma Miyake ◽  
Jun Kawamoto ◽  
Yun-Lin Wei ◽  
Masanari Kitagawa ◽  
Ikunoshin Kato ◽  
...  
Keyword(s):  
Low Temperature ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Maximum Yield ◽  
Broad Host Range ◽  
Psychrophilic Bacterium ◽  
T7 Promoter ◽  
Cold Adapted ◽  
Protein Expression System

ABSTRACT A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4°C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce β-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4°C and 139 mg/liter of culture at 18°C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.

scholarly journals Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
B. Karsten Tischer ◽  
Benedikt B. Kaufer
Keyword(s):  
Dna Sequences ◽  
Rna Virus ◽  
Virus Genome ◽  
Virus Species ◽  
Bacterial Artificial Chromosomes ◽  
Nucleotide Substitutions ◽  
Recombinant Viruses ◽  
Artificial Chromosomes ◽  
Dna And Rna ◽  
Virus Genomes

Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques inEscherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members ofCoronaviridaeandFlaviviridaecould be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process.

scholarly journals Codon Preference Optimization Increases Prokaryotic Cystatin C Expression

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Qing Wang ◽  
Cui Mei ◽  
Honghua Zhen ◽  
Jess Zhu
Keyword(s):  
Protein Expression ◽  
Cystatin C ◽  
Production Systems ◽  
Codon Optimization ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Gc Content ◽  
Optimization Techniques ◽  
E Coli ◽  
Prokaryotic Expression Vector

Gene expression is closely related to optimal vector-host system pairing in many prokaryotes. Redesign of the humancystatin C(cysC) gene using the preferred codons of the prokaryotic system may significantly increasecysCexpression inEscherichia coli(E. coli). Specifically,cysCexpression may be increased by removing unstable sequences and optimizing GC content. According toE. coliexpression system codon preferences, the gene sequence was optimized while the amino acid sequence was maintained. The codon-optimizedcysC(co-cysC) and wild-typecysC(wt-cysC) were expressed by cloning the genes into a pET-30a plasmid, thus transforming the recombinant plasmid intoE. coliBL21. Before and after the optimization process, the prokaryotic expression vector and host bacteria were examined for protein expression and biological activation of CysC. The recombinant proteins in the lysate of the transformed bacteria were purified using Ni2+-NTA resin. Recombinant protein expression increased from 10% to 46% based on total protein expression after codon optimization. Recombinant CysC purity was above 95%. The significant increase incysCexpression inE. coliexpression produced by codon optimization techniques may be applicable to commercial production systems.

scholarly journals Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e12497
Author(s):  
Fei Shang ◽  
Hui Wang ◽  
Dan Zhang ◽  
Wenhui Wang ◽  
Jiangliu Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Protein Expression ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Promoter Regions ◽  
T7 Promoter ◽  
Iptg Induction ◽  
Pet Expression System

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

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