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Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

ABSTRACT: This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesized protein was enhanced by as much as 10 times in the presence of an optimal second codon. The signal peptides were in situ cleaved and the target proteins were produced in their native sizes by carrying out the cell-free synthesis reactions in the presence of Triton X-100, most likely through the activation of signal peptidase in the S30 extract. The amplification of the template DNA and the addition of the signal sequences were accomplished by PCR. Hence, elevated levels of recombinant proteins were generated within several hours.

SUBMITTER: Ahn JH

PROVIDER: S-EPMC1849898 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

Ahn Jin-Ho JH Hwang Mi-Yeon MY Lee Kyung-Ho KH Choi Cha-Yong CY Kim Dong-Myung DM

Nucleic acids research 20061220 4

This study developed a method to boost the expression of recombinant proteins in a cell-free protein synthesis system without leaving additional amino acid residues. It was found that the nucleotide sequences of the signal peptides serve as an efficient downstream box to stimulate protein synthesis when they were fused upstream of the target genes. The extent of stimulation was critically affected by the identity of the second codons of the signal sequences. Moreover, the yield of the synthesize ...[more]

PMID: 17185295

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Project description:Purpose:Heterophoria describes the deviation of the optical axes in the absence of binocular fusion. Eye trackers (ET) can provide an objective assessment but are not broadly used clinically. We examined the feasibility of combining an infrared (IR) pass-filter, IR detector, and an off-the-shelf ET. The proposed setup was validated against the broadly used cover test (CT). Furthermore, the setup was used to examine whether testing conditions can affect the measurements. Methods:An IR detector was attached to a handheld IR-pass filter that blocks visible light to provide occlusion while passing IR light for eye tracking. The detector senses the IR illumination of the eye tracker, creating a recordable signal of the occluder position synchronized with eye positions acquired by the SMI Red250 tracker. The mean of three measurements of each condition, three versus ten seconds occlusion, the occluded eye, and ET versus CT results were compared using the Wilcoxon test, correlation and Bland and Altman plots. Differences between measurements that were within 2? were considered clinically insignificant. Results:Thirty normally-sighted subjects (mean age 24.50 ± 2.20, range 20-28) with heterophoria ranging between 14? exophoria and 4? esophoria were recruited. There was no significant difference between the occluded eyes. However, there was a difference between 3 and 10 seconds' cover duration. The CT data were more similar to the 10 seconds cover duration, although differences were less than the clinical resolution of 2?. Conclusions:An inexpensive off-the-shelf ET can be used to measure heterophoria with controlled testing parameters. Translational Relevance:Our study demonstrated a robust technique for synchronization of an optical element such as an IR cover, with an off-the-shelf commercial eye tracker. The synchronization of optical elements with eye tracking, which has been described here for heterophoria, can be adapted for other clinical measurements.

Dataset Information

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

Publications

Use of signal sequences as an in situ removable sequence element to stimulate protein synthesis in cell-free extracts.

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