Understanding heterologous protein overproduction under the T7 promoter: A practical exercise
نویسندگان
چکیده
منابع مشابه
Gene 18 protein of bacteriophage T7. Overproduction, purification, and characterization.
Genetic and physical analyses indicate that gene 18 protein of bacteriophage T7 is essential for packaging of T7 DNA. T7 DNA is replicated via linear intermediates, culminating in the formation of concatemers many genomes in length which are then packaged into capsids. In infections with phage carrying amber mutations in gene 18, development is blocked at the concatemer stage. Biochemical studi...
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Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Ad...
متن کاملLow-copy-number T7 vectors for selective gene expression and efficient protein overproduction in Escherichia coli.
A set of low-copy-number vectors (pPD) has been constructed that permit selective gene expression and high-level protein overproduction in Escherichia coli, based on the bacteriophage T7 RNA polymerase/T7 promoter system. These plasmids carry a chloramphenicol resistance gene (cat) as a selective marker and an extended multiple cloning site for convenient gene cloning. Their replication is medi...
متن کاملSuperior 5' homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter.
Transcription from the commonly used GTP- initiating T7 class III promoter phi6.5 frequently produces heterogeneous RNA at both 3' and 5' ends. We demonstrate here that RNA transcripts from the T7 class II promoter phi2.5 have superior 5' homogeneity over those from the phi6.5 promoter, with comparable total RNA yields. The overall homogeneity of RNA transcripts is improved to different degrees...
متن کاملPromoter specificity determinants of T7 RNA polymerase.
The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage ...
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ژورنال
عنوان ژورنال: Biochemistry and Molecular Biology Education
سال: 2002
ISSN: 1470-8175,1539-3429
DOI: 10.1002/bmb.2002.494030030066