TAKARA Single Protein Production

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Single Protein Production system (SPP systemTM). This system utilizes an E. coli protein MazF, 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. 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.

A Single Protein Production system (SPP system TM ) 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 A Single Protein Production system (SPP system TM ) 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 TM . 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 expres- sion, 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 (Figure3) which is ready to express MazF in adequate quantity. The transformant with the both plasmids generates the SPP of interest. Takara’s SPP System ineludes 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), 6*His-Tag, and Factor Xa cleavage site (table & Figure4). pColdIV (SP-4) has no additional sequence at the upstream of the MCS. same group and it was refered to as an mRNA Interferase TM . The protein was deter- mined 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  equences. 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 expres- sion, 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 (Figure3) which is ready to express MazF in adequate quantity. The transformant with the both plasmids generates the SPP of interest. Takara’s SPP System ineludes 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), 6*His-Tag, and Factor Xa cleavage site (table & Figure4). pColdIV (SP-4) has no additional sequence at the upstream of the MCS.

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