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Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

Bottom Line: Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis.Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library.The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

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Reporter proteins MXB and RtcB characterization on SDS-PAGE. (A) The MXB protein (from NEB pMXB plasmid) is a fusion of an N-terminal MBP, linked through the Mycobacterium xenopi gyrA mini-intein to the CBP. In the presence of DTT, the intein mediates peptide cleavage into the two domains. (B) Three MXB variants of C384, S384 and UAG384 differ in the intein-cleavage active site. After Ni-NTA purification in the absence of reducing agents, DTT was added to a final concentration of 50 mM and aliquots were taken at the indicated time points. The cleavage patterns for the MXB variants are shown in the Coomassie Blue stained SDS-PAGE. (C) The Pyrococcus horikoshii RtcB C98, S98 and UAG98 variants are expressed with the UTu system in the absence or presence of the indicated EF-Tu variants for subsequent Ni-NTA purification. Aliquots of the Ni-NTA eluate are separated through SDS-PAGE and analyzed by western blot against the carboxyterminal His6-tag. The positions of intact RtcB C98/S98 proteins and the cleaved RtcB U98 fragment are indicated.
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Figure 5: Reporter proteins MXB and RtcB characterization on SDS-PAGE. (A) The MXB protein (from NEB pMXB plasmid) is a fusion of an N-terminal MBP, linked through the Mycobacterium xenopi gyrA mini-intein to the CBP. In the presence of DTT, the intein mediates peptide cleavage into the two domains. (B) Three MXB variants of C384, S384 and UAG384 differ in the intein-cleavage active site. After Ni-NTA purification in the absence of reducing agents, DTT was added to a final concentration of 50 mM and aliquots were taken at the indicated time points. The cleavage patterns for the MXB variants are shown in the Coomassie Blue stained SDS-PAGE. (C) The Pyrococcus horikoshii RtcB C98, S98 and UAG98 variants are expressed with the UTu system in the absence or presence of the indicated EF-Tu variants for subsequent Ni-NTA purification. Aliquots of the Ni-NTA eluate are separated through SDS-PAGE and analyzed by western blot against the carboxyterminal His6-tag. The positions of intact RtcB C98/S98 proteins and the cleaved RtcB U98 fragment are indicated.

Mentions: We tested our UTu system for Sec incorporation with four different proteins. The first two examples are of intein-mediated protein splicing. The first case was the control plasmid pMXB10 of the New England Biolabs IMPACT system. In this plasmid the coding region of the maltose-binding protein (MBP) is fused to the Mxe gyrase intein–chitin binding domain. Upon incubation of the full-length 70 kDa protein with DTT, the intein active site C384 cleaves the adjacent peptide bond to produce two fragments of MBP (42 kDa) and intein–chitin binding protein (CBP, 28 kDa). A similar cleavage is seen by the U384-containing protein; no significant cleavage activity has been seen for MXB S384 (Figure 5A). Judged by the cleavage pattern Cys and Sec are equally active.


Engineering the elongation factor Tu for efficient selenoprotein synthesis.

Haruna K, Alkazemi MH, Liu Y, Söll D, Englert M - Nucleic Acids Res. (2014)

Reporter proteins MXB and RtcB characterization on SDS-PAGE. (A) The MXB protein (from NEB pMXB plasmid) is a fusion of an N-terminal MBP, linked through the Mycobacterium xenopi gyrA mini-intein to the CBP. In the presence of DTT, the intein mediates peptide cleavage into the two domains. (B) Three MXB variants of C384, S384 and UAG384 differ in the intein-cleavage active site. After Ni-NTA purification in the absence of reducing agents, DTT was added to a final concentration of 50 mM and aliquots were taken at the indicated time points. The cleavage patterns for the MXB variants are shown in the Coomassie Blue stained SDS-PAGE. (C) The Pyrococcus horikoshii RtcB C98, S98 and UAG98 variants are expressed with the UTu system in the absence or presence of the indicated EF-Tu variants for subsequent Ni-NTA purification. Aliquots of the Ni-NTA eluate are separated through SDS-PAGE and analyzed by western blot against the carboxyterminal His6-tag. The positions of intact RtcB C98/S98 proteins and the cleaved RtcB U98 fragment are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Reporter proteins MXB and RtcB characterization on SDS-PAGE. (A) The MXB protein (from NEB pMXB plasmid) is a fusion of an N-terminal MBP, linked through the Mycobacterium xenopi gyrA mini-intein to the CBP. In the presence of DTT, the intein mediates peptide cleavage into the two domains. (B) Three MXB variants of C384, S384 and UAG384 differ in the intein-cleavage active site. After Ni-NTA purification in the absence of reducing agents, DTT was added to a final concentration of 50 mM and aliquots were taken at the indicated time points. The cleavage patterns for the MXB variants are shown in the Coomassie Blue stained SDS-PAGE. (C) The Pyrococcus horikoshii RtcB C98, S98 and UAG98 variants are expressed with the UTu system in the absence or presence of the indicated EF-Tu variants for subsequent Ni-NTA purification. Aliquots of the Ni-NTA eluate are separated through SDS-PAGE and analyzed by western blot against the carboxyterminal His6-tag. The positions of intact RtcB C98/S98 proteins and the cleaved RtcB U98 fragment are indicated.
Mentions: We tested our UTu system for Sec incorporation with four different proteins. The first two examples are of intein-mediated protein splicing. The first case was the control plasmid pMXB10 of the New England Biolabs IMPACT system. In this plasmid the coding region of the maltose-binding protein (MBP) is fused to the Mxe gyrase intein–chitin binding domain. Upon incubation of the full-length 70 kDa protein with DTT, the intein active site C384 cleaves the adjacent peptide bond to produce two fragments of MBP (42 kDa) and intein–chitin binding protein (CBP, 28 kDa). A similar cleavage is seen by the U384-containing protein; no significant cleavage activity has been seen for MXB S384 (Figure 5A). Judged by the cleavage pattern Cys and Sec are equally active.

Bottom Line: Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis.Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library.The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Show MeSH
Related in: MedlinePlus