SILVER
SILVER ๋“ฑ๊ธ‰์˜ ํŒ๋งค์ž ์ž๋ฃŒ

[A+]์„œ๊ฐ•๋Œฟ’ํ•™๊ป“ ํ˜„๋Œ€์ƒ๋ฌผํ•™์”„ํ—˜1_2์ฐจ ํ’€๋ ˆํฌํŠธ_6x His tagged eGFP๋ฐœํ˜„ ํ›„ ๋‹จ๋ฐฑ์งˆ ์ •์ œ ๋ฐ ํš๋“, ๋‹จ๋ฐฑ์งˆ ํ™•์ธ ๋ฐ ์ •๋Ÿ‰์œผ๋กœ eGFP ์ˆœ๋„ ํ™•์ธ

17 ํŽ˜์ด์งฟ’
์›Œ๋“œ
์ตœ์ดˆ๋“ฑ๋ก์ผ 2025.02.23 ์ตœ์ข…์ ฟ’์ž‘์ผ 2024.04
17P ๋ฏธ๋้ฉ๋ณด๊ธฐ
[A+]์„œ๊ฐ•๋Œฟ’ํ•™๊ป“ ํ˜„๋Œ€์ƒ๋ฌผํ•™์”„ํ—˜1_2์ฐจ ํ’€๋ ˆํฌํŠธ_6x His tagged eGFP๋ฐœํ˜„ ํ›„ ๋‹จ๋ฐฑ์งˆ ์ •์ œ ๋ฐ ํš๋“, ๋‹จ๋ฐฑ์งˆ ํ™•์ธ ๋ฐ ์ •๋Ÿ‰์œผ๋กœ eGFP ์ˆœ๋„ ํ™•์ธ
  • ๋ฏธ๋้ฉ๋ณด๊ธฐ

    ์†Œ๊ฐœ

    "[A+]์„œ๊ฐ•๋Œฟ’ํ•™๊ป“ ํ˜„๋Œ€์ƒ๋ฌผํ•™์”„ํ—˜1_2์ฐจ ํ’€๋ ˆํฌํŠธ_6x His tagged eGFP๋ฐœํ˜„ ํ›„ ๋‹จ๋ฐฑ์งˆ ์ •์ œ ๋ฐ ํš๋“, ๋‹จ๋ฐฑ์งˆ ํ™•์ธ ๋ฐ ์ •๋Ÿ‰์œผ๋กœ eGFP ์ˆœ๋„ ํ™•์ธ"์— ๋Œ€ํ•œ ๋‚ด์šฉ์ž…๋‹ˆ๋‹ค.

    ๋ชฉ์ฐจ

    Abstract
    Introduction
    Materials & Methods
    Results
    Discussion
    References

    ๋ณธ๋ๅฉ๋‚ด์šฉ

    EGFP๋Š” ํ•ดํŒŒ๋ฆฌ Aequorea victoria์—์„œ ๋ฐœ๊ฒฌ๋œ GFP๊ฐ€ ๋ณ€ํ˜•๋œ ํ˜•ํƒœ๋กœ 488nm์—์„œ excitation์ด ์ผ์–ด๋‚˜๋„ 509nm์—์„œ ํ˜•๊ด‘์„ ๋ฐฉ์ถœํ•œ๋‹ค. Reporter gene์œผ๋กœ ์‚ฌ์šฉ๋˜๋ฉฐ, Target gene์˜ ๋ฐœํ˜„ ์ •๋„๋ฅผ ํ™•์ธํ•˜๊ณ , ์„ธํฌ ๋‚ด์—์„œ ๋‹จ๋ฐฑ์งˆ์˜ ๋ฐœํ˜„ ๋ถ€์œ„๋ฅผ ๊ด€์ฐฐํ•œ๋‹ค. EGFP๋ฅผ ๋ฐœํ˜„์‹œ์ผœ ์ •์ œํ•œ ๋’ค ํš๋“ํ•˜์—ฌ ์ •๋Ÿ‰ ๊ณผ์ •์„ ๊ฑฐ์ณค๋‹ค. ๋ณธ ์‹คํ—˜์—์„œ eGFP๋ฅผ ์–ป๊ธฐ ์œ„ํ•ด 6x His-tagged eGFP gene์ด ์‚ฝ์ž…๋˜์–ด ์žˆ๋Š” pET28a(+)๋ฅผ ์ด์šฉํ•˜์˜€๊ณ , BL21(DE3)์— transformationํ•˜์˜€๋‹ค. T7 RNA polymerase๋ฅผ lac operon system์— IPTG๋ฅผ ์ด์šฉํ•˜์—ฌ ์ง€์†์ ์œผ๋กœ induction์‹œํ‚ค๊ณ , 6x His-tagged eGFP๋ฅผ ๊ณผ๋ฐœํ˜„ ์‹œ์ผฐ๋‹ค. Immobilized metal ion affinity chromatography๋ฅผ ์ด์šฉํ•˜์—ฌ Histidine๊ณผ Ni2+-NTA resin๊ณผ ํŠน์ด์ ์œผ๋กœ ๊ฒฐํ•ฉํ•˜์—ฌ ๋ชฉ์  ๋‹จ๋ฐฑ์งˆ์„ ์ˆœ์ˆ˜ ์ •์ œํ•˜์˜€๊ณ , Amicon centrifugal fulter tube๋ฅผ ์ด์šฉํ•˜์—ฌ ultrafiltration์„ ์ง„ํ–‰ํ–ˆ๋‹ค. SDS-PAGE๋ฅผ ํ†ตํ•ด ์ •์ œํ•œ ๋‹จ๋ฐฑ์งˆ์˜ ๋ถ„์ž๋Ÿ‰์— ๋”ฐ๋ฅธ band๋ฅผ ํ™•์ธํ•˜์˜€์œผ๋ฉฐ, whole lysate๋‚˜ Flow through๋Š” ์—ฌ๋Ÿฌ ์œ„์น˜์—์„œ band๊ฐ€ ๊ด€์ฐฐ๋˜๋Š” ๋ฐ˜๋ฉด, ์ตœ์ข… ์ •์ œ eGFP์˜ ๊ฒฝ์šฐ 28.3 kDa ์œ„์น˜์—๋งŒ ๋‘๊บผ์šด band๊ฐ€ ํ™•์ธ๋˜์–ด ์ •์ œ๊ฐ€ ์ž˜ ์ด๋ฃจ์–ด ์ง์„ ์•Œ ์ˆ˜ ์žˆ๋‹ค. 280nm, 488nm์—์„œ Extinction coefficient assay๋ฅผ ํ•˜์—ฌ ๊ตฌํ•œ eGFP์˜ ๋†๋„๋Š” ๊ฐ๊ฐ 0.298mM, 0.199mM๋กœ total eGFP, functional eGFP ๋†๋„๋ฅผ ๋‚˜ํƒ€๋‚ธ๋‹ค. Lowry assay๋กœ ๊ตฌํ•œ eGFP์˜ ์งˆ๋Ÿ‰๋†๋„๋Š” BSA๋ฅผ ์ด์šฉํ•˜์—ฌ standard curve๋ฅผ ๊ทธ๋ ค ์–ป์–ด๋‚ธ ๊ฐ’์œผ๋กœ, 13.78mg/ml์ด๋ฉฐ, ๋ชฐ๋†๋„๋Š” 0.487mM์ด๋‹ค.

    ์ฐธ๊ณ ์ž๋ฃŒ

    ยท Spriestersbach, A., Kubicek, J., Schรคfer, F., Block, H., & Maertens, B. (2015). Purification of his-tagged proteins. In Methods in enzymology, Vol. 559, pp. 1-15
    ยท Gill, S. C., & Von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Analytical biochemistry, 182(2), 319-326
    ยท Pieลกลฅanskรฝ, Juraj & Galba, Jaroslav & Oleลกovรก, Dominika & Kovacech, Branislav & Kovac, Andrej. (2019). Determination of immunogenic proteins in biopharmaceuticals by UHPLCโ€“MS amino acid analysis. BMC Chemistry. 13. 10.1186/s13065-019-0581-z.
    ยท Raran-Kurussi, S., Cherry, S., Zhang, D., & Waugh, D. S. (2017). Removal of affinity tags with TEV protease. Heterologous Gene Expression in E. coli: Methods and Protocols, pp. 221-230
    ยท Schรคfer, F., Seip, N., Maertens, B., Block, H., & Kubicek, J. (2015). Purification of GST-tagged proteins. In Methods in enzymology, Vol. 559, pp. 127-139
    ยท 6. Jenny, R. J., Mann, K. G., & Lundblad, R. L. (2003). A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein expression and purification, 31(1), 1-11
    ยท Kowit, J. D., & Maloney, J. (1982). Protein cleavage by boiling in sodium dodecyl sulfate prior to electrophoresis. Analytical Biochemistry, 123(1), 86-93
    ยท Kruger, N. J. (2009). The Bradford method for protein quantitation. The protein protocols handbook, pp. 17-24
    ยท Walker, J. M. (2009). The bicinchoninic acid (BCA) assay for protein quantitation. The protein protocols handbook, pp. 11-15
    ยท Dave Nelson, Michael Cox (2021) Lehninger principles of biochemistry, eighth edition, W. H. Freeman, pp.402-409
    ยท David P. Clark and Nanette J. Pazdernik (2016), Biotechnology, 2nd ed, Elsevier, pp.311-313
  • Easy Ai ์š”์•ฝ

    ์ด ๋ฌธ์„œ๋Š” 6x His-tagged eGFP ๋ฐœํ˜„ ๋ฐ ์ •์ œ ์‹คํ—˜ ๊ฒฐ๊ณผ๋ฅผ ๋ณด์—ฌ์ค€๋‹ค. IPTG ์œ ๋„ ํ•˜์— BL21(DE3) ๋Œ€์žฅ๊ท ์—์„œ 6x His-tagged eGFP๋ฅผ ๊ณผ๋ฐœํ˜„์‹œํ‚ค๊ณ , Ni-NTA affinity chromatography์™ฟ’ ultrafiltration์„ ํ†ตํ•ด ๋‹จ๋ฐฑ์งˆ์„ ์ •์ œํ•˜์˜€๋‹ค. SDS-PAGE ๋ถ„์„ ๊ฒฐ๊ณผ ์ตœ์ข… ์ •์ œ ๋‹จ๋ฐฑ์งˆ์—์„œ ๋‹จ์ผ ๋ฐด๋“œ๋ฅผ ํ™•์ธํ•˜์˜€์œผ๋ฉฐ, UV-Vis ๋ถ„๊ด‘๊ด‘๋„๊ณ„์™ฟ’ Lowry assay๋ฅผ ์ด์šฉํ•˜์—ฌ eGFP์˜ ๋†๋„์™ฟ’ ์ˆœ๋„๋ฅผ ์ •๋Ÿ‰ํ•˜์˜€๋‹ค. Spectrofluorometer ๋ถ„์„์„ ํ†ตํ•ด eGFP์˜ ๊ณ ์œ ํ•œ excitation ๋ฐ emission ํŒŒ์žฅ์„ ํ™•์ธํ•˜์˜€๋‹ค. ๋ณธ ์‹คํ—˜์€ ๋‹จ๋ฐฑ์งˆ ๋ฐœํ˜„, ์ •์ œ, ํŠน์„ฑ ๋ถ„์„์— ํ™œ์šฉ๋˜๋Š” ๋‹ค์–‘ํ•œ ๊ธฐ๋ฒ•๋“ค์„ ์ฒด๊ณ„์ ์œผ๋กœ ๋ณด์—ฌ์ฃผ๋ฉฐ, ์ •์ œ๋œ eGFP ๋‹จ๋ฐฑ์งˆ์˜ ์ˆœ๋„์™ฟ’ ๊ธฐ๋Šฅ์„ฑ์„ ์ข…ํ•ฉ์ ์œผ๋กœ ๊ฒ€์ฆํ•˜์˜€๋‹ค. ๋”ฐ๋ผ์„œ ์ด ๋ฌธ์„œ๋Š” eGFP์™ฟ’ ๊ฐ™์€ ํ˜•๊ด‘ ๋‹จ๋ฐฑ์งˆ์˜ ๋ฐœํ˜„ ๋ฐ ์ •์ œ ๊ณผ์ •์„ ์ดํ•ดํ•˜๊ณ  ๋ถ„์„ํ•˜๋Š”๋ฐ ๋„์›€์„ ์ค„ ๊ฒƒ์œผ๋กœ ํŒ๋‹จ๋œ๋‹ค.
  • ์ž๋ฃŒํ›„๊ธฐ

      Ai ๋ฆฌ๋ทฐ
      ๋ณธ ์‹คํ—˜์€ eGFP์˜ ๋ฐœํ˜„, ์ •์ œ, ์ •๋Ÿ‰ ๊ณผ์ •์„ ์ฒด๊ณ„์ ์œผ๋กœ ์ˆ˜ํ–‰ํ•˜์˜€์œผ๋ฉฐ, ๋‹ค์–‘ํ•œ ๋ถ„์„ ๊ธฐ๋ฒ•์„ ํ™œ์šฉํ•˜์—ฌ ๋‹จ๋ฐฑ์งˆ์˜ ์ˆœ๋„์™ฟ’ ํŠน์„ฑ์„ ์ข…ํ•ฉ์ ์œผ๋กœ ํ™•์ธํ•˜์˜€๋‹ค.
    • ์ž์ฃผ๋ฌป๋Š”์งˆ๋ๅฉ์˜ ๋‹ต๋ณ€์„ ํ™•์ธํ•ด ์ฃผ์„ธ์š”

      ํ•ดํ”ผ์บ ํผ์Šค FAQ ๋”๋ต–๊ธฐ

      ๊ผญ ์•Œ์•„์ฃผ์„ธ์š”

      • ์ž๋ฃŒ์˜ ์ •๋ณด ๋ฐ ๋‚ด์šฉ์˜ ์ง„์‹ค์„ฑ์— ๋Œ€ํ•˜์—ฌ ํ•ดํ”ผ์บ ํผ์Šค๋Š” ๋ณด์ฆํ•˜์ง€ ์•Š์œผ๋ฉฐ, ํ•ด๋‹น ์ •๋ณด ๋ฐ ๊ฒŒ์‹œ๋ฌผ ์ €์ž‘๊ถŒ๊ณผ ๊ธฐํƒ€ ๋ฒ•์  ์ฑ…์ž„์€ ์ž๋ฃŒ ๋“ฑ๋ก์ž์—๊ฒŒ ์žˆ์Šต๋‹ˆ๋‹ค.
        ์ž๋ฃŒ ๋ฐ ๊ฒŒ์‹œ๋ฌผ ๋‚ด์šฉ์˜ ๋ถˆ๋ฒ•์  ์ด์šฉ, ๋ฌด๋‹จ ์ „์žฌโˆ™๋ฐฐํฌ๋Š” ๊ธˆ์ง€๋˜์–ด ์žˆ์Šต๋‹ˆ๋‹ค.
        ์ €์ž‘๊ถŒ์นจํ•ด, ๋ช…์˜ˆํ›ผ์† ๋“ฑ ๋ถ„์Ÿ ์š”์†Œ ๋ฐœ๊ฒฌ ์‹œ ๊ณ ๊ฐ๋น„๋ฐ”์นด์ง€๋…ธ Viva์˜ ์ €์ž‘๊ถŒ์นจํ•ด ์‹ ๊ณ ๋น„๋ฐ”์นด์ง€๋…ธ Viva๋ฅผ ์ด์šฉํ•ด ์ฃผ์‹œ๊ธฐ ๋ฐ”๋ž๋‹ˆ๋‹ค.
      • ํ•ดํ”ผ์บ ํผ์Šค๋Š” ๊ตฌ๋งค์ž์™ฟ’ ํŒ๋งค์ž ๋ชจ๋‘๊ฐ€ ๋งŒ์กฑํ•˜๋Š” ์„œ๋น„์Šค๊ฐ€ ๋˜๋„๋ก ๋…ธ๋ ฅํ•˜๊ณ  ์žˆ์œผ๋ฉฐ, ์•„๋ž˜์˜ 4๊ฐ€์ง€ ์ž๋ฃŒํ™˜๋ถˆ ์กฐ๊ฑด์„ ๊ผญ ํ™•์ธํ•ด์ฃผ์‹œ๊ธฐ ๋ฐ”๋ž๋‹ˆ๋‹ค.
        ํŒŒ์ผ์˜ค๋ฅ˜ ์ค‘๋ณต์ž๋ฃŒ ์ €์ž‘๊ถŒ ์—†์Œ ์„ค๋ช…๊ณผ ์‹ค์ œ ๋‚ด์šฉ ๋ถˆ์ผ์น˜
        ํŒŒ์ผ์˜ ๋‹ค์šด๋กœ๋“œ๊ฐ€ ์ œ๋Œ€๋กœ ๋˜์ง€ ์•Š๊ฑฐ๋‚˜ ํŒŒ์ผํ˜•์‹์— ๋งž๋Š” ํ”„๋กœ๊ทธ๋žจ์œผ๋กœ ์ •์ƒ ์ž‘๋™ํ•˜์ง€ ์•Š๋Š” ๊ฒฝ์šฐ ๋‹ค๋ฅธ ์ž๋ฃŒ์™ฟ’ 70% ์ด์ƒ ๋‚ด์šฉ์ด ์ผ์น˜ํ•˜๋Š” ๊ฒฝ์šฐ (์ค‘๋ณต์ž„์„ ํ™•์ธํ•  ์ˆ˜ ์žˆ๋Š” ๊ทผ๊ฑฐ ํ•„์š”ํ•จ) ์ธํ„ฐ๋„ท์˜ ๋‹ค๋ฅธ ์‚ฌ์ดํŠธ, ์—ฐ๊ตฌ๊ธฐ๊ด€, ํ•™๊ป“, ์„œ์  ๋“ฑ์˜ ์ž๋ฃŒ๋ฅผ ๋„์šฉํ•œ ๊ฒฝ์šฐ ์ž๋ฃŒ์˜ ์„ค๋ช…๊ณผ ์‹ค์ œ ์ž๋ฃŒ์˜ ๋‚ด์šฉ์ด ์ผ์น˜ํ•˜์ง€ ์•Š๋Š” ๊ฒฝ์šฐ
    ๋ฌธ์„œ ์ดˆ์•ˆ์„ ์ƒ์„ฑํ•ด์ฃผ๋Š” EasyAI
    ์•ˆ๋…•ํ•˜์„ธ์š”. ํ•ดํ”ผ์บ ํผ์Šค์˜ ๋ฐฉ๋Œ€ํ•œ ์ž๋ฃŒ ์ค‘์—์„œ ์„ ๋ณ„ํ•˜์—ฌ ๋‹น์‹ ๋งŒ์˜ ์ดˆ์•ˆ์„ ๋งŒ๋“ค์–ด์ฃผ๋Š” EasyAI ์ž…๋‹ˆ๋‹ค.
    ์ €๋Š” ์•„๋ž˜์™ฟ’ ๊ฐ™์ด ์ž‘์—…์„ ๋„์™ฟ’๋“œ๋ฆฝ๋‹ˆ๋‹ค.
    - ์ฃผ์ œ๋งŒ ์ž…๋ ฅํ•˜๋ฉด ๋ชฉ์ฐจ๋ถ€ํ„ฐ ๋ณธ๋ๅฉ๋‚ด์šฉ๊นŒ์ง€ ์ž๋™ ์ƒ์„ฑํ•ด ๋“œ๋ฆฝ๋‹ˆ๋‹ค.
    - ์žฅ๋ฌธ์˜ ์ฝ˜ํ…์ธ ๋ฅผ ์‰ฝ๊ณ  ๋น ๋ฅด๊ฒŒ ์ž‘์„ฑํ•ด ๋“œ๋ฆฝ๋‹ˆ๋‹ค.
    - ์Šคํ† ์–ด์—์„œ ๋ฌด๋ฃŒ ์บ์‹œ๋ฅผ ๊ณ„์ •๋ณ„๋กœ 1ํšŒ ๋ฐœ๊ธ‰ ๋ฐ›์„ ์ˆ˜ ์žˆ์Šต๋‹ˆ๋‹ค. ์ง€๊ธˆ ๋ฐ”๋กœ ์ฒดํ—˜ํ•ด ๋ณด์„ธ์š”!
    ์ด๋Ÿฐ ์ฃผ์ œ๋“ค์„ ์ž…๋ ฅํ•ด ๋ณด์„ธ์š”.
    - ์œ ์•„์—๊ฒŒ ์ ํ•ฉํ•œ ๋ฌธํ•™์ž‘ํ’ˆ์˜ ๊ธฐ์ค€๊ณผ ํŠน์„ฑ
    - ํ•œ๊ตญ์ธ์˜ ๊ฐ€์น˜๊ด€ ์ค‘์—์„œ ์ •์‹ ์  ๊ฐ€์น˜๊ด€์„ ์ด๋ฃจ๋Š” ๊ฒƒ๋“ค์„ ๋ฌธํ™”์  ๋ฌธ๋ฒ•์œผ๋กœ ์ •๋ฆฌํ•˜๊ณ , ํ˜„๋Œ€ํ•œ๊ตญ์‚ฌํšŒ์—์„œ ์ผ์–ด๋‚˜๋Š” ์‚ฌ๊ฑด๊ณผ ์‚ฌ๊ณ ๋ฅผ ๋น„๊ตํ•˜์—ฌ ์ž์‹ ์˜ ์˜๊ฒฌ์œผ๋กœ ๊ธฐ์ˆ ํ•˜์„ธ์š”
    - ์ž‘๋ณ„์ธ์‚ฌ ๋…ํ›„๊ฐ
    ํ•ด์บ  AI ์ฑ—๋ด‡๊ณผ ๋Œ€ํ™”ํ•˜๊ธฐ
    ์ฑ—๋ด‡์œผ๋กœ ๊ฐ„ํŽธํ•˜๊ฒŒ ์ƒ๋‹ดํ•ด๋ณด์„ธ์š”.
    2025๋…„ 06์›” 22์ผ ์ผ์š”์ผ
    AI ์ฑ—๋ด‡
    ์•ˆ๋…•ํ•˜์„ธ์š”. ํ•ดํ”ผ์บ ํผ์Šค AI ์ฑ—๋ด‡์ž…๋‹ˆ๋‹ค. ๋ฌด์—‡์ด ๊ถ๊ธˆํ•˜์‹ ๊ฐ€์š”?
    7:01 ์˜ค์ „