Products >> Proteomics >> Protein Expression >> SPP System™ (Single Protein Production System) - Best suitable for isotope labeling
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Description

A Single Protein Production system (SPP system™) was proposed by Dr. M. Inouye's group at Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey. This system utilizes an E. coli protein MazF which was found by the same group and it was refered to as an mRNA Interferase™. The protein was determined to be a sequence-specific endoribonuclease which cleaves single strand RNAs at ACA sequences. In this system, the transcript of interest which should not contain any ACA sequences (i.e. ACA-less), and MazF are co-expressed in a host Escherichia coli. Therefore the MazF does not cleave the transcript of interest, but cleave the ones derived from the host proteins or others at ACA sequences. So, only the transcript of interest is dominantly translated and only the protein of interest is dominantly expressed, with the SPP (Figure1). Instead of their dominant expression, some proteins of interest are expressed in lesser amount with this system than in other expression systems, for example, in Cold-shock expression vector system alone.
In order to construct SPP system™, it is first necessary to prepare an ACA-less gene of interest by chemical synthesis or site-directed mutagenesis technology, etc. In designing the ACA-less gene, every ACA sequence in the gene of interest must be substituted to another keeping its amino acid sequence, and restriction sites for cloning must be also added to be in frame. Secondly the ACA-less gene is cloned into a ACA-less region of the pCold™ (SP-4) vector which has ACA-less transcription region. At this point the plasmid becomes ready for SPP of interest. Then a host E. coli is co-transformed with this expression plasmid for SPP and pMazF which is ready to express MazF in adequate quantity. The transformant with the both plasmids generates the SPP of interest.
Takara's SPP System™ includes four types of Cold-shock expression vectors for SPP, pCold™ I (SP-4), pCold™ II (SP-4), pCold™ III (SP-4) and pCold™ IV (SP-4), which vary in the existences of TEE (translation enhancing element), His-Tag, and Factor Xa cleavage site (table). pCold™ IV (SP-4) has no additional sequence at the upstream of the MCS.

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Components

Cat.# 3366
 

	Cold Shock Expression vector for SPP System™ (0.5 µg/µl) pCold™ I (SP-4) DNA, pCold™ II (SP-4) DNA, pCold™ III (SP-4) DNA, pCold™ IV (SP-4) DNA	ea. 20 µg
	MazF (mRNA Interferase™) expression plasmid pMazF DNA (20 ng/µl)	0.5 µg
	Positive Control pCold™ I (SP-4) envZB DNA* (20 ng/µl)	0.2 µg

* Expression plasmid prepared by inserting ORF of E. coli-derived protein envZB without ACA sequence into pCold™ I (SP-4) DNA Estimated molecular weight of expressed protein 19.6 kDa

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SPP System™ I (Cat.# 3367)

	Cold Shock Expression vector for SPP System™ pCold™ I (SP-4) DNA	20 µg (0.5 µg/µl)
	MazF (mRNA Interferase™) expression plasmid pMazF DNA	0.5 µg (20 ng/µl)
	Positive Control pCold™ I (SP-4) envZB DNA	0.2 µg (20 ng/µl)

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SPP System™ II (Cat.# 3368)

	Cold Shock Expression vector for SPP System™ pCold™ II (SP-4) DNA	20 µg (0.5 µg/µl)
	MazF (mRNA Interferase™) expression plasmid pMazF DNA	0.5 µg (20 ng/µl)
	Positive Control pCold™ I (SP-4) envZB DNA	0.2 µg (20 ng/µl)

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SPP System™ III (Cat.# 3369)

	Cold Shock Expression vector for SPP System™ pCold™ III (SP-4) DNA	20 µg (0.5 µg/µl)
	MazF (mRNA Interferase™) expression plasmid pMazF DNA	0.5 mg (20 ng/µl)
	Positive Control pCold™ I (SP-4) envZB DNA	0.2 µg (20 ng/µl)

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SPP System™ IV (Cat.# 3370)

	Cold Shock Expression vector for SPP System™ pCold™ IV (SP-4) DNA	20 µg (0.5 µg/µl)
	MazF (mRNA Interferase™) expression plasmid pMazF DNA	0.5 µg (20 ng/µl)
	Positive Control pCold™ I (SP-4) envZB DNA	0.2 µg (20 ng/µl)

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Principle of SPP System

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Features

	Convenient for radioisotope labeling Up to 90%, at maximum, of newly expressed cellular protein is labeled target protein.
	Wide range of E. coli hosts Most E. coli strains are available since cspA promoter derived from E. coli is utilized. However, pMazF DNA used for co-expression utilizes chloramphenicol as a selection marker, chloramphenicol-resistant E. coli strain, e.g. Rossetta™ (Novagen) cannot be used as a host.

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GenBank

	Accession No.
pCold I(SP-4)	AB248600
pCold II(SP-4)	AB248601
pCold III(SP-4)	AB248602
pCold IV(SP-4)	AB248603

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Vector Map

	TEE sequence*	His-Tag sequence	Factor Xa cleavage site
pCold™ I (SP-4) DNA	Ο	Ο	Ο
pCold™ II (SP-4) DNA	Ο	Ο	×
pCold™ III (SP-4) DNA	Ο	×	×
pCold™ IV (SP-4) DNA	×	×	×

* TEE : translation enhancing element

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