Catching up to nature's ribosomes

doi:10.1126/science.abb9711

GSTDTAP > 气候变化

DOI	10.1126/science.abb9711
	Catching up to nature's ribosomes
	Caroline Proulx
	2020-05-29
发表期刊	Science
出版年	2020
英文摘要	Chemical protein synthesis allows the systematic incorporation of one or more unnatural amino acids at precise locations along a protein backbone that enable structure-function relationship studies ([ 1 ][1], [ 2 ][2]). However, solid-phase peptide synthesis (SPPS) ([ 3 ][3]) is usually limited to peptides of 50 residues, and as a result, chemical protein synthesis requires native chemical ligation (NCL) ([ 4 ][4]) reactions for the chemoselective coupling of unprotected peptide fragments in solution. This combined multistep approach typically requires the synthesis and sequential ligation of at least three peptide fragments to access proteins, a process that remains relatively laborious. On page 980 of this issue, Hartrampf et al. ([ 5 ][5]) chemically synthesized single-domain proteins in hours. They circumvent the use of solution-phase chemistry by carefully optimizing iterative amino acid couplings on a solid support using a flow-chemistry setup (see the figure). The introduction of SPPS in the 1960s ([ 3 ][3]) revolutionized peptide sciences. The attachment of the carboxyl-terminal amino acid to an insoluble polymeric support allowed each reaction to be done with excess reagents that could be easily washed off through simple filtration, a technique that earned Bruce Merrifield the Nobel Prize in Chemistry in 1984. Because there are no purifications steps in between each amino acid coupling and deprotection reactions performed on a solid support, near-quantitative yields are required for each monomer addition cycle to enable the synthesis and purification of proteins. Common by-products that have plagued peptide syntheses have included those arising from deletions, truncations, aspartimide formation, and amino acid epimerizations. Each unwanted reaction yields material that is often difficult or impossible to separate from the desired product with high-performance liquid chromatography purification methods. Although much progress has been made to address these challenges ([ 6 ][6]), peptide sequence lengths that can be routinely accessed with traditional SPPS approaches fall well below the lengths of entire protein sequences. This limitation has required the development and use of postsynthetic ligation methods. However, a wide variety of unnatural amino acids can be incorporated using this approach. Alternatively, incorporation of unnatural amino acids into proteins can be done through genetic code expansion in biological systems instead ([ 7 ][7]), albeit with differences in substrate scope. In a previous study from this team, Mijalis et al. reported automated fast-flow peptide synthesis (AFPS) procedures that substantially accelerated the process and improves the synthesis of difficult peptide sequences ([ 8 ][8]). Notably, their AFPS system has shortened the amino acid addition cycle to less than 1 min, and convenient inline ultraviolet-visible monitoring has allowed quantitative analysis of the fluorenylmethoxycarbonyl (Fmoc) deprotection reactions. This feature offers the marked advantage of identifying problematic coupling steps rapidly and consistently throughout the synthesis, versus the time-consuming manual analyses typically used. ![Figure][9] Fast-flow automated protein synthesis Hartrampf et al. optimized concentration, flow rate, reagents, additives, and temperature to minimize unwanted by-products in peptide synthesis. The peptide lengths are in the range of those of single-domain proteins. GRAPHIC: N. CARY/ SCIENCE ; (STRUCTURES) HARTRAMPF ET AL . ( 5 ) In the present study, Hartrampf et al. have methodically optimized the parameters of their AFPS setup, including concentration, flow rate, reagents, additives, and temperature, to surmount the difficult task of reducing by-products sufficiently to enable protein synthesis ([ 5 ][5]). They established particular sets of optimal conditions for each amino acid incorporation by first using the 30-residue glucagon-like peptide-1 as a test. They then applied their optimized procedure to the synthesis of nine different proteins, thus establishing a generalized protocol for total chemical protein synthesis by AFPS. Comparative analyses with proteins produced by biological expression validated that the synthetic proteins obtained in this study exhibited similar structure and function. Overall, in addition to its impressive speed, this new technique leads to a three-fold increase in peptide size limit achievable by AFPS techniques. This advance should facilitate future protein synthesis endeavors and influence multiple fields of research. Coupling AFPS technology with existing NCL methods could deliver >300-residue proteins after a single ligation reaction. Moreover, it is easy to imagine similar optimization efforts for a wide variety of unnatural amino acid residues, expanding their utility far beyond their current scope and potentially yielding all-unnatural protein–mimetic material that is inaccessible by biological methods. In that respect, it will not only catch up to but surpass ribosome capabilities. 1. [↵][10]1. S. B. H. Kent , Chem. Soc. Rev. 38, 338 (2009). [OpenUrl][11][CrossRef][12][PubMed][13][Web of Science][14] 2. [↵][15]1. V. Agouridas et al ., Chem. Rev. 119, 7328 (2019). [OpenUrl][16][CrossRef][17][PubMed][18] 3. [↵][19]1. R. B. Merrifield , J. Am. Chem. Soc. 85, 2149 (1963). [OpenUrl][20][CrossRef][21][Web of Science][22] 4. [↵][23]1. P. E. Dawson, 2. T. W. Muir, 3. I. Clark-Lewis, 4. S. B. Kent , Science 266, 776 (1994). [OpenUrl][24][Abstract/FREE Full Text][25] 5. [↵][26]1. N. Hartrampf et al ., Science 368, 980 (2020). [OpenUrl][27][Abstract/FREE Full Text][28] 6. [↵][29]1. R. Behrendt, 2. P. White, 3. J. Offer , J. Pept. Sci. 22, 4 (2016). [OpenUrl][30][CrossRef][31][PubMed][32] 7. [↵][33]1. L. Wang, 2. P. G. Schultz , Angew. Chem. Int. Ed. 44, 34 (2004). [OpenUrl][34][CrossRef][35][PubMed][36][Web of Science][37] 8. [↵][38]1. A. J. Mijalis et al ., Nat. Chem. Biol. 13, 464 (2017). [OpenUrl][39][CrossRef][40][PubMed][41] Acknowledgments: The author gratefully acknowledges North Carolina State University for startup support. 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领域	气候变化 ; 资源环境
URL	查看原文
引用统计
文献类型	期刊论文
条目标识符	http://119.78.100.173/C666/handle/2XK7JSWQ/271746
专题	气候变化资源环境科学
推荐引用方式 GB/T 7714	Caroline Proulx. Catching up to nature's ribosomes[J]. Science,2020.
APA	Caroline Proulx.(2020).Catching up to nature's ribosomes.Science.
MLA	Caroline Proulx."Catching up to nature's ribosomes".Science (2020).