5 research outputs found

    Structural dissection of human translation elongation factor 1BΞ³ (eEF1BΞ³): expression of full-length protein and its truncated forms

    No full text
    Aim. To gain more insights into properties of the human translation elongation factor eEF1BΞ³ and its interaction with partners we intended to produce the full-length protein and its truncated forms. Methods. cDNAs encoding truncated forms of eEF1BΞ³ were generated by PCR amplification with respective primers and cloned into vectors providing polyhistidine, glutathione S-transferase or maltose binding protein tags. The recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography. An aggregation state of the proteins was analyzed by analytical gel filtration. Results. The expression, purification and storage conditions for the full-length recombinant His-eEF1BΞ³ were optimized. Several truncated forms of eEF1BΞ³ were also expressed and purified to homogeneity. Two short variants of C-terminal domain comprising amino acids 263–437 or 228–437 were obtained in monomeric state. Two short variants of N-terminal domain comprising amino acids 1–33 or 1–230, fused with glutathione S-transferase, were obtained and estimated to be dimers by gel filtration. The mutants of N-terminal domain comprising amino acids 1–93 or 1–165, fused with maltose binding protein, were obtained as soluble high molecular weight aggregates only. Conclusions. The purified recombinant His-eEF1BΞ³ and several truncated forms of the protein were obtained and characterized. These protein variants will be used for further studies on the protein-protein interaction.ΠœΠ΅Ρ‚Π°. Для Π΄Π΅Ρ‚Π°Π»ΡŒΠ½ΠΎΠ³ΠΎ вивчСння властивостСй Ρ„Π°ΠΊΡ‚ΠΎΡ€Π° Π΅Π»ΠΎΠ½Π³Π°Ρ†Ρ–Ρ— трансляції eEF1BΞ³ людини Ρ– ΠΉΠΎΠ³ΠΎ Π²Π·Π°Ρ”ΠΌΠΎΠ΄Ρ–Ρ— Π· ΠΏΠ°Ρ€Ρ‚Π½Π΅Ρ€Π°ΠΌΠΈ ΠΎΠΏΡ‚ΠΈΠΌΡ–Π·ΡƒΠ²Π°Ρ‚ΠΈ Π΅ΠΊΡΠΏΡ€Π΅ΡΡ–ΡŽ ΠΊΠ”ΠΠš ΠΏΠΎΠ²Π½ΠΎΡ€ΠΎΠ·ΠΌΡ–Ρ€Π½ΠΎΠ³ΠΎ Π±Ρ–Π»ΠΊΠ° Ρ‚Π° ΠΉΠΎΠ³ΠΎ Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½ΠΈΡ… Ρ„ΠΎΡ€ΠΌ. ΠœΠ΅Ρ‚ΠΎΠ΄ΠΈ. ΠΊΠ”ΠΠš, які ΠΊΠΎΠ΄ΡƒΡŽΡ‚ΡŒ Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Ρ– Ρ„ΠΎΡ€ΠΌΠΈ eEF1BΞ³, синтСзували ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ ΠŸΠ›Π -Π°ΠΌΠΏΠ»Ρ–Ρ„Ρ–ΠΊΠ°Ρ†Ρ–Ρ— Π· Π²Ρ–Π΄ΠΏΠΎΠ²Ρ–Π΄Π½ΠΈΠΌΠΈ ΠΏΡ€Π°ΠΉΠΌΠ΅Ρ€Π°ΠΌΠΈ Ρ– ΠΊΠ»ΠΎΠ½ΡƒΠ²Π°Π»ΠΈ Ρƒ Π²Π΅ΠΊΡ‚ΠΎΡ€ΠΈ, Ρ‰ΠΎ ΠΌΡ–ΡΡ‚ΡΡ‚ΡŒ які Π°Ρ„Ρ–Π½Π½Ρƒ ΠΌΡ–Ρ‚ΠΊΡƒ полігістидинову ΠΏΠΎΡΠ»Ρ–Π΄ΠΎΠ²Π½Ρ–ΡΡ‚ΡŒ, Π³Π»ΡƒΡ‚Π°Ρ‚Ρ–ΠΎΠ½ S-трансфСразу Π°Π±ΠΎ Π±Ρ–Π»ΠΎΠΊ, який зв’язує ΠΌΠ°Π»ΡŒΡ‚ΠΎΠ·Ρƒ. Π Π΅ΠΊΠΎΠΌΠ±Ρ–Π½Π°Π½Ρ‚Π½Ρ– Π±Ρ–Π»ΠΊΠΈ СкспрСсували Π² бактСріях Ρ– ΠΎΡ‡ΠΈΡ‰ΡƒΠ²Π°Π»ΠΈ Π°Ρ„Ρ–Π½Π½ΠΎΡŽ Ρ…Ρ€ΠΎΠΌΠ°Ρ‚ΠΎΠ³Ρ€Π°Ρ„Ρ–Ρ”ΡŽ. АгрСгатний стан ΠΎΡ‚Ρ€ΠΈΠΌΠ°Π½ΠΈΡ… Π±Ρ–Π»ΠΊΡ–Π² Π°Π½Π°Π»Ρ–Π·ΡƒΠ²Π°Π»ΠΈ Π· використанням Π°Π½Π°Π»Ρ–Ρ‚ΠΈΡ‡Π½ΠΎΡ— гСль-Ρ„Ρ–Π»ΡŒΡ‚Ρ€Π°Ρ†Ρ–Ρ—. Π Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚ΠΈ. ΠžΠΏΡ‚ΠΈΠΌΡ–Π·ΠΎΠ²Π°Π½ΠΎ Π΅ΠΊΡΠΏΡ€Π΅ΡΡ–ΡŽ, очищСння Ρ‚Π° ΡƒΠΌΠΎΠ²ΠΈ збСрігання ΠΏΠΎΠ²Π½ΠΎΡ€ΠΎΠ·ΠΌΡ–Ρ€Π½ΠΎΠ³ΠΎ Ρ€Π΅ΠΊΠΎΠΌΠ±Ρ–Π½Π°Π½Ρ‚Π½ΠΎΠ³ΠΎ His-eEF1BΞ³. Π’Π°ΠΊΠΎΠΆ СкспрСсовано Ρ– ΠΎΡ‡ΠΈΡ‰Π΅Π½ΠΎ Π΄ΠΎ Π³ΠΎΠΌΠΎΠ³Π΅Π½Π½ΠΎΠ³ΠΎ стану ΠΊΡ–Π»ΡŒΠΊΠ° Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½ΠΈΡ… Ρ„ΠΎΡ€ΠΌ eEF1BΞ³. Π”Π²Ρ– Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Ρ– Ρ„ΠΎΡ€ΠΌΠΈ Π‘-ΠΊΡ–Π½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Ρƒ, які ΠΌΡ–ΡΡ‚ΡΡ‚ΡŒ амінокислотні залишки 263–437 Ρ– 228–437, ΠΎΡ‚Ρ€ΠΈΠΌΠ°Π½ΠΎ Ρƒ вигляді Ρ€ΠΎΠ·Ρ‡ΠΈΠ½Π½ΠΈΡ… ΠΌΠΎΠ½ΠΎΠΌΠ΅Ρ€Π½ΠΈΡ… Π±Ρ–Π»ΠΊΡ–Π². Π”Π²Ρ– Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Ρ– Ρ„ΠΎΡ€ΠΌΠΈ N-ΠΊΡ–Π½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Ρƒ, Ρ‰ΠΎ ΠΌΡ–ΡΡ‚ΡΡ‚ΡŒ амінокислотні залишки 1–33 Ρ– 1–230, Π·Π»ΠΈΡ‚Ρ– Π· Π³Π»ΡƒΡ‚Π°Ρ‚Ρ–ΠΎΠ½ S-Ρ‚Ρ€Π°Π½ΡΡ„Π΅Ρ€Π°Π·ΠΎΡŽ, ΠΎΠ΄Π΅Ρ€ΠΆΠ°Π½ΠΎ Ρƒ вигляді Π΄ΠΈΠΌΠ΅Ρ€Ρ–Π² Π·Π³Ρ–Π΄Π½ΠΎ Π· Ρ€Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Π°ΠΌΠΈ гСль-Ρ„Ρ–Π»ΡŒΡ‚Ρ€Π°Ρ†Ρ–Ρ—. Π†Π½ΡˆΡ– Π΄Π΅Π»Π΅Ρ†Ρ–ΠΉΠ½Ρ– ΠΌΡƒΡ‚Π°Π½Ρ‚ΠΈ, які ΠΌΡ–ΡΡ‚ΡΡ‚ΡŒ амінокислотні залишки 1–93 Ρ– 1–165 N-ΠΊΡ–Π½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Ρƒ Ρ‚Π° Π·Π»ΠΈΡ‚Ρ– Π· Π±Ρ–Π»ΠΊΠΎΠΌ, Ρ‰ΠΎ зв’язує ΠΌΠ°Π»ΡŒΡ‚ΠΎΠ·Ρƒ, ΠΌΠΎΠΆΡƒΡ‚ΡŒ Π±ΡƒΡ‚ΠΈ ΠΎΡ‚Ρ€ΠΈΠΌΠ°Π½Ρ– лишС Ρƒ вигляді Ρ€ΠΎΠ·Ρ‡ΠΈΠ½Π½ΠΈΡ… високомолСкулярних Π°Π³Ρ€Π΅Π³Π°Ρ‚Ρ–Π². Висновки. ΠžΡ‚Ρ€ΠΈΠΌΠ°Π½ΠΎ Ρ– ΠΎΡ…Π°Ρ€Π°ΠΊΡ‚Π΅Ρ€ΠΈΠ·ΠΎΠ²Π°Π½ΠΎ Ρ€Π΅ΠΊΠΎΠΌΠ±Ρ–Π½Π°Π½Ρ‚Π½ΠΈΠΉ Π±Ρ–Π»ΠΎΠΊ His-eEF1BΞ³ Ρ‚Π° ΠΉΠΎΠ³ΠΎ Ρ‡ΠΎΡ‚ΠΈΡ€ΠΈ Π²ΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Ρ– Ρ„ΠΎΡ€ΠΌΠΈ. Π¦Ρ– Ρ„ΠΎΡ€ΠΌΠΈ Π² ΠΏΠΎΠ΄Π°Π»ΡŒΡˆΠΎΠΌΡƒ Π±ΡƒΠ΄Π΅ використано для вивчСння Π±Ρ–Π»ΠΊΠΎΠ²ΠΎ-Π±Ρ–Π»ΠΊΠΎΠ²ΠΈΡ… Π²Π·Π°Ρ”ΠΌΠΎΠ΄Ρ–ΠΉ.ЦСль. Для Π±ΠΎΠ»Π΅Π΅ Π΄Π΅Ρ‚Π°Π»ΡŒΠ½ΠΎΠ³ΠΎ изучСния свойств чСловСчСского Ρ„Π°ΠΊΡ‚ΠΎΡ€Π° элонгации трансляции eEF1BΞ³ ΠΈ Π΅Π³ΠΎ взаимодСйствия с ΠΏΠ°Ρ€Ρ‚Π½Π΅Ρ€Π°ΠΌΠΈ ΠΎΠΏΡ‚ΠΈΠΌΠΈΠ·ΠΈΡ€ΠΎΠ²Π°Ρ‚ΡŒ Π΅ΠΊΡΠΏΡ€Π΅ΡΡΠΈΡŽ ΠΊΠ”ΠΠš ΠΏΠΎΠ»Π½ΠΎΡ€Π°Π·ΠΌΠ΅Ρ€Π½ΠΎΠ³ΠΎ Π±Π΅Π»ΠΊΠ°, Π° Ρ‚Π°ΠΊΠΆΠ΅ Π΅Π³ΠΎ усСчСнных Ρ„ΠΎΡ€ΠΌ. ΠœΠ΅Ρ‚ΠΎΠ΄Ρ‹. ΠΊΠ”ΠΠš, ΠΊΠΎΠ΄ΠΈΡ€ΡƒΡŽΡ‰ΠΈΠ΅ ΡƒΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Π½Ρ‹Π΅ Ρ„ΠΎΡ€ΠΌΡ‹ eEF1BΞ³, Π³Π΅Π½Π΅Ρ€ΠΈΡ€ΠΎΠ²Π°Π»ΠΈ ΠΌΠ΅Ρ‚ΠΎΠ΄ΠΎΠΌ ПЦР-Π°ΠΌΠΏΠ»ΠΈΡ„ΠΈΠΊΠ°Ρ†ΠΈΠΈ с ΡΠΎΠΎΡ‚Π²Π΅Ρ‚ΡΡ‚Π²ΡƒΡŽΡ‰ΠΈΠΌΠΈ ΠΏΡ€Π°ΠΉΠΌΠ΅Ρ€Π°ΠΌΠΈ ΠΈ ΠΊΠ»ΠΎΠ½ΠΈΡ€ΠΎΠ²Π°Π»ΠΈ Π² Π²Π΅ΠΊΡ‚ΠΎΡ€Ρ‹, содСрТащиС Π² качСствС Π°Ρ„Ρ„ΠΈΠ½Π½ΠΎΠΉ ΠΌΠ΅Ρ‚ΠΊΠΈ ΠΏΠΎΠ»ΠΈΠ³ΠΈΡΡ‚ΠΈΠ΄ΠΈΠ½ΠΎΠ²ΡƒΡŽ ΠΏΠΎΡΠ»Π΅Π΄ΠΎΠ²Π°Ρ‚Π΅Π»ΡŒΠ½ΠΎΡΡ‚ΡŒ, Π³Π»ΡƒΡ‚Π°Ρ‚ΠΈΠΎΠ½ S-трансфСразу ΠΈΠ»ΠΈ Π±Π΅Π»ΠΎΠΊ, ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰ΠΈΠΉ ΠΌΠ°Π»ΡŒΡ‚ΠΎΠ·Ρƒ. Π Π΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½Ρ‚Π½Ρ‹Π΅ Π±Π΅Π»ΠΊΠΈ экспрСссировали Π² бактСриях ΠΈ ΠΎΡ‡ΠΈΡ‰Π°Π»ΠΈ Π°Ρ„Ρ„ΠΈΠ½Π½ΠΎΠΉ Ρ…Ρ€ΠΎΠΌΠ°Ρ‚ΠΎΠ³Ρ€Π°Ρ„ΠΈΠ΅ΠΉ. АгрСгатноС состояниС ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Π½Ρ‹Ρ… Π±Π΅Π»ΠΊΠΎΠ² Π°Π½Π°Π»ΠΈΠ·ΠΈΡ€ΠΎΠ²Π°Π»ΠΈ с ΠΏΠΎΠΌΠΎΡ‰ΡŒΡŽ аналитичСской гСль-Ρ„ΠΈΠ»ΡŒΡ‚Ρ€Π°Ρ†ΠΈΠΈ. Π Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Ρ‹. ΠžΠΏΡ‚ΠΈΠΌΠΈΠ·ΠΈΡ€ΠΎΠ²Π°Π½Ρ‹ экспрСссия, очистка ΠΈ условия хранСния ΠΏΠΎΠ»Π½ΠΎΡ€Π°Π·ΠΌΠ΅Ρ€Π½ΠΎΠ³ΠΎ Ρ€Π΅ΠΊΠΎΠΌΠ±ΠΈ- Π½Π°Π½Ρ‚Π½ΠΎΠ³ΠΎ His-eEF1BΞ³. Π’Π°ΠΊΠΆΠ΅ СкспрСссированы ΠΈ ΠΎΡ‡ΠΈΡ‰Π΅Π½Ρ‹ Π΄ΠΎ Π³ΠΎΠΌΠΎΠ³Π΅Π½Π½ΠΎΠ³ΠΎ состояния нСсколько усСчСнных Ρ„ΠΎΡ€ΠΌ eEF1BΞ³. Π”Π²Π΅ ΡƒΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Π½Ρ‹Π΅ Ρ„ΠΎΡ€ΠΌΡ‹ Π‘-ΠΊΠΎΠ½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Π°, содСрТащиС аминокис- Π»ΠΎΡ‚Π½Ρ‹Π΅ остатки 263–437 ΠΈ 228–437, ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Ρ‹ Π² Π²ΠΈΠ΄Π΅ растворимых ΠΌΠΎΠ½ΠΎΠΌΠ΅Ρ€Π½Ρ‹Ρ… Π±Π΅Π»ΠΊΠΎΠ². Π”Π²Π΅ ΡƒΠΊΠΎΡ€ΠΎΡ‡Π΅Π½Π½Ρ‹Π΅ Ρ„ΠΎΡ€ΠΌΡ‹ N-ΠΊΠΎΠ½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Π°, содСрТащиС аминокислотныС остатки 1–33 ΠΈ 1–230, слитыС с Π³Π»ΡƒΡ‚Π°Ρ‚ΠΈΠΎΠ½ S-трансфСразой, ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Ρ‹ Π² Π²ΠΈΠ΄Π΅ Π΄ΠΈΠΌΠ΅Ρ€ΠΎΠ² ΠΏΠΎ Ρ€Π΅Π·ΡƒΠ»ΡŒΡ‚Π°Ρ‚Π°ΠΌ гСль-Ρ„ΠΈΠ»ΡŒΡ‚Ρ€Π°Ρ†ΠΈΠΈ. Π”Ρ€ΡƒΠ³ΠΈΠ΅ Π΄Π΅Π»Π΅Ρ†ΠΈΠΎΠ½Π½Ρ‹Π΅ ΠΌΡƒΡ‚Π°Π½Ρ‚Ρ‹, содСрТащиС аминокислотныС остатки 1–93 ΠΈ 1–165 N-ΠΊΠΎΠ½Ρ†Π΅Π²ΠΎΠ³ΠΎ Π΄ΠΎΠΌΠ΅Π½Π° ΠΈ слиты с Π±Π΅Π»ΠΊΠΎΠΌ, ΡΠ²ΡΠ·Ρ‹Π²Π°ΡŽΡ‰ΠΈΠΌ ΠΌΠ°Π»ΡŒΡ‚ΠΎΠ·Ρƒ, ΠΌΠΎΠ³ΡƒΡ‚ Π±Ρ‹Ρ‚ΡŒ ΠΏΠΎΠ»ΡƒΡ‡Π΅Π½Ρ‹ Ρ‚ΠΎΠ»ΡŒΠΊΠΎ Π² Π²ΠΈΠ΄Π΅ растворимых высокомолСкулярных Π°Π³Ρ€Π΅Π³Π°Ρ‚ΠΎΠ². Π’Ρ‹Π²ΠΎΠ΄Ρ‹. ΠŸΠΎΠ»ΡƒΡ‡Π΅Π½Ρ‹ ΠΈ ΠΎΡ…Π°Ρ€Π°ΠΊΡ‚Π΅Ρ€ΠΈΠ·ΠΎΠ²Π°Π½Ρ‹ Ρ€Π΅ΠΊΠΎΠΌΠ±ΠΈΠ½Π°Π½Ρ‚Π½Ρ‹ΠΉ Ρ„Π°ΠΊΡ‚ΠΎΡ€ элонгации трансляции His-eEF1B ΠΈ Π΅Π³ΠΎ Ρ‡Π΅Ρ‚Ρ‹Ρ€Π΅ усСчСнныС Ρ„ΠΎΡ€ΠΌΡ‹, ΠΊΠΎΡ‚ΠΎΡ€Ρ‹Π΅ Π² дальнСйшСм Π±ΡƒΠ΄ΡƒΡ‚ ΠΈΡΠΏΠΎΠ»ΡŒΠ·ΠΎΠ²Π°Π½Ρ‹ для ΠΈΠ·Ρƒ- чСния Π±Π΅Π»ΠΊΠΎΠ²ΠΎ-Π±Π΅Π»ΠΊΠΎΠ²Ρ‹Ρ… взаимодСйствий

    Profiles de microhabitats dans des hΓͺtraies primaires d'Europe

    No full text
    International audienceTree-related microhabitats (TreMs) are important features for the conservation of biodiversity in forest ecosystems. Although other structural indicators of forest biodiversity have been extensively studied in recent decades, TreMs have often been overlooked, either due to the absence of a consensual definition or a lack of knowledge. Despite the increased number of TreM studies in the last decade, the role of drivers of TreM profile in primary forests and across different geographical regions is still unknown. To evaluate the main drivers of TreM density and diversity, we conducted the first large-scale study of TreMs across European primary forests. We established 146 plots in eight primary forests dominated by European beech (Fagus sylvatica L.) in the Carpathian and Dinaric mountain ranges. Generalized linear mixed effect models were used to test the effect of local plot characteristics and spatial variability on the density and diversity (alpha, beta, and gamma) of TreMs. Total TreM density and diversity were significantly positively related with tree species richness and the proportion of snags. Root mean square tree diameters were significantly related to alpha and gamma diversity of TreMs. Both regions reached similarly high values of total TreM densities and total TreM densities and diversity were not significantly different between the two regions; however, we observed between the two regions significant differences in the densities of two TreM groups, conks of fungi and epiphytes. The density and diversity of TreMs were very high in beech-dominated mountain primary forests, but their occurrence and diversity was highly variable within the landscapes over relatively short spatial gradients (plot and stand levels). Understanding these profile provides a benchmark for further comparisons, such as with young forest reserves, or for improving forest management practices that promote biodiversity
    corecore