Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.
Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.
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DOI：
10.1016/j.jbc.2022.101846
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Gould, Stephen J.
中科院分区：
文献类型：
--
作者：
Tsai, Shang Jui;Ai, Yiwei;Guo, Chenxu;Gould, Stephen J.
5个抗生素抗性(AR)基因已用于筛选转基因真核细胞系，其中BleoR、PuroR、HygR、NeoR和BsdR盒分别对玉米素、腐霉素、潮霉素、遗传素/G418和杀菌素产生抗性。我们最近证明，每个AR基因都建立了一个不同的转基因表达阈值，低于这个阈值，任何细胞都不能存活，BleoR选择转基因表达水平最高的细胞，∼几乎是使用neoR或BsdR标记选择的细胞的10倍。在这里，我们测试了一种假设，即AR蛋白功能与连接的、转基因编码的重组蛋白的表达之间可能存在相反的比例。具体地说，我们将每个AR蛋白融合到蛋白酶体靶向degron标签，使用这些标签来选择抗生素耐药细胞系，然后测量连接的重组蛋白mCherry的表达，作为转基因表达的代理标记。在每一种情况下，变性标记的AR蛋白都选择了比其同源的WT AR蛋白更高的mCherry表达。ER50BleoR被选为mCherry表达水平最高的基因，比BleoR或任何其他AR基因高出两倍以上。有趣的是，使用ER50BleoR作为可选择的标记，可以使胞外体货物蛋白CD63/Y235A的胞外体负载量增加3.5倍。尽管一种可能的CD63结合肽CP05已经被用来装饰外体膜，在这里我们发现CP05与CD63−/−细胞、WT293F细胞和CD63过表达细胞同样能很好地结合，这表明CP05可能是非特异性的结合膜。这些结果对细胞工程，特别是外切体工程具有重要意义。
Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as “exosome painting,” we show here that CP05 binds equally well to CD63−/− cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.
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选择标记的选择会影响细胞和外泌体中的重组蛋白表达。
DOI：
10.1016/j.jbc.2021.100838
发表时间：
2021-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Guo C;Fordjour FK;Tsai SJ;Morrell JC;Gould SJ
通讯作者：
Gould SJ
影响因子：
14.9
作者：
KASTER, KR;BURGETT, SG;INGOLIA, TD
通讯作者：
INGOLIA, TD
影响因子：
64.5
作者：
BOSHART, M;WEBER, F;SCHAFFNER, W
通讯作者：
SCHAFFNER, W
DOI：
10.1073/pnas.76.9.4317
发表时间：
1979-01-01
影响因子：
11.1
作者：
GRUSS, P;LAI, CJ;KHOURY, G
通讯作者：
KHOURY, G
DOI：
10.1073/pnas.79.5.1573
发表时间：
1982-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
JOYNER, A;YAMAMOTO, Y;BERNSTEIN, A
通讯作者：
BERNSTEIN, A