​pHIS1525

pHIS1525

pHIS1525载体基本信息：

pHIS1525载体质粒图谱和多克隆位点信息：

pHIS1525载体简介：

Plasmids pHIS1522/pHIS1525 both contain the strong xylA promoter (PxylA)

originating from Bacillus megaterium. Transcription from this promoter is xylose

inducible (after xylose addition, the xylose repressor coded by the xylR gene on

the plasmids is released from PxylA and transcription is initiated). The most

convenient cloning sites for insertion of DNA fragments carrying heterologous

genes are located in an open reading frame (orf1) under control of the xyloseinducible

promoter PxylA (15 unique restriction sites; see sequence). Therefore, any

protein can be expressed using one out of three functionally different fusion

strategies.

A transcriptional fusion requires that the gene of interest carries its own ribosome

binding sequence (RBS) and translation initiation codon. Such a DNA fragment

can be fused into any of the available restriction sites within orf1. Whether the

resulting transcriptional fusion leads to expression of the gene of interest, which

is independent from orf1 expression, depends on the location of the created orf1

stop codon with respect to the start codon of the gene of interest. lf these are close

together, translational coupling may occur, in which the ribosomes translating the

orf1 reading frame would terminate at its stop codon, creating a locally high

concentration of ribosomes, so that translation initiation at the new start codon

would be more efficient compared to a construct in which the orf1 translation

terminates farther away from the start codon.

On the other hand, if the orf1 reading frame continues for a long distance into the

reading frame of the gene of interest, the ribosomes translating the created orf1

fusion protein might inhibit initiation of translation of the protein of interest.

Therefore, it is advisable to pay attention to placement of a stop codon when

constructing the gene fusion. Taken together, although a transcriptional or operon

fusion is constructed, the efficient translation of the orf1 reading frame, and any

fusion thereof created by the insertion, is likely to, positively or negatively,

influence the translation efficiency of the gene of interest.

Alternatively, a truncated version of the gene of interest, lacking its own start

codon, may be fused in frame to the orf1 reading frame on pHIS1522/pHIS1525

to create a translational or protein fusion. This will result in expression of a chimeric

protein consisting of up to 18 amino acids specified by the orf1 encoding

sequence, followed by the sequence encoded by the gene of interest.

Using the BsrGI restriction site directly before the ATG start codon enables cloning

without changing the N-terminus of the protein of interest. pHIS1525 contains a

sequence encoding the B. subtilis extracellular esterase (LipA) and allows cloning

of target genes directly after the signal peptidase restriction site using the Kas I,

Nar I, or SfoI restriction sites.

It is important to note that the multiple cloning site and its reading frame are

identical in pHIS1522 and pHIS1525 starting from BglII. Hence, a parallel

cloning strategy of the gene of interest in pHIS1522 for intracellular and in

pHIS1525 for extracellular production is possible. The 6xHis sequence upstream

the MCS allows convenient purification and detection of the expressed Histagged

target proteins. For expression of target proteins without a 6xHis tag,

suitable plasmid constructs (pMM1522 & pMM1525) are also available.

pHIS1525载体其他相关的其他类型载体：