Name: overexpression and purification of human cis-prenyltransferase in escherichia coli
Uploaded: 2017-08-03T15:00:00
Description: Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Dette arbejde blev finansieret af Israels Videnskabsstiftelsens Center for Forskning Excellence (I-CORE) i Strukturel Cellbiologi (1775/12) og Israels Videnskabsstiftelse tildeler 1721/16 og 2338/16 (YH) og 825/14 (DK ). Støtten fra Fields Estate Foundation til DK er meget værdsat. Dette arbejde blev udført af Ilan Edri og Michal Goldenberg i delvis opfyldelse af MD-afhandlingskravene fra Sackler Fakultet for Medicin, Tel Aviv Universitet.

1. Kloning af cis -PT til overekspression i E. coli <ol><li> Hent pET-32b ekspressionsvektor, der er designet til kloning og ekspression på højt niveau af proteinsekvenser fusioneret med 109aa thioredoxin (TRX) protein 17 og E. coli kodon-optimeret 18 kodende sekvens af cis -PT med fuld længde. </li><li> Pas på at have et spaltningssted for TEV-protease (tobakse etchvirusprotease) (ENLYFQ / G, hvor &quot;/&quot; angiver spaltningsp...

<ol>
	<li>Grabinska, K. A., Park, E. J., Sessa, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis-Prenyltransferase:+New+Insights+into+Protein+Glycosylation,+Rubber+Synthesis,+and+Human+Diseases.">cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases.</a> J Biol Chem. 291 (35), 18582-18590 (2016).</li><li>Ogura, K., Koyama, T., Sagami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyprenyl+diphosphate+synthases.">Polyprenyl diphosphate synthases.</a> Subcell Biochem. 28, 57-87 (1997).</li><li>Ogura, K., Koyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Aspects+of+Isoprenoid+Chain+Elongation.">Enzymatic Aspects of Isoprenoid Chain Elongation.</a> Chem Rev. 98 (4), 1263-1276 (1998).</li><li>Imperiali, B., O'Connor, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+N-linked+glycosylation+on+glycopeptide+and+glycoprotein+structure.">Effect of N-linked glycosylation on glycopeptide and glycoprotein structure.</a> Curr Opin Chem Biol. 3 (6), 643-649 (1999).</li><li>Bukhtiyarov, Y. E., Shabalin, Y. A., Kulaev, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+and+characterization+of+dehydrodolichyl+diphosphate+synthase+from+the+yeast+Saccharomyces+carlsbergensis.">Solubilization and characterization of dehydrodolichyl diphosphate synthase from the yeast Saccharomyces carlsbergensis.</a> J Biochem. 113 (6), 721-728 (1993).</li><li>Adair, W. L., Cafmeyer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Saccharomyces+cerevisiae+cis-prenyltransferase+required+for+dolichyl+phosphate+biosynthesis.">Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis.</a> Arch Biochem Biophys. 259 (2), 589-596 (1987).</li><li>Zelinger, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+missense+mutation+in+DHDDS,+encoding+dehydrodolichyl+diphosphate+synthase,+is+associated+with+autosomal-recessive+retinitis+pigmentosa+in+Ashkenazi+Jews.">A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in Ashkenazi Jews.</a> Am J Hum Genet. 88 (2), 207-215 (2011).</li><li>Zuchner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-exome+sequencing+links+a+variant+in+DHDDS+to+retinitis+pigmentosa.">Whole-exome sequencing links a variant in DHDDS to retinitis pigmentosa.</a> Am J Hum Genet. 88 (2), 201-206 (2011).</li><li>Tropea, J. E., Cherry, S., Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+soluble+His(6)-tagged+TEV+protease.">Expression and purification of soluble His(6)-tagged TEV protease.</a> Methods Mol Biol. 498, 297-307 (2009).</li><li>Crouse, G. F., Frischauf, A., Lehrach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+and+simplified+approach+to+cloning+into+plasmids+and+single-stranded+phages.">An integrated and simplified approach to cloning into plasmids and single-stranded phages.</a> Methods Enzymol. 101, 78-89 (1983).</li><li>Middelberg, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process-scale+disruption+of+microorganisms.">Process-scale disruption of microorganisms.</a> Biotechnol Adv. 13 (3), 491-551 (1995).</li><li>Block, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilized-metal+affinity+chromatography+(IMAC):+a+review.">Immobilized-metal affinity chromatography (IMAC): a review.</a> Methods Enzymol. 463, 439-473 (2009).</li><li>Sachyani, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+a+Kv7.1+potassium+channel+gating+module:+studies+of+the+intracellular+c-terminal+domain+in+complex+with+calmodulin.">Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.</a> Structure. 22 (11), 1582-1594 (2014).</li><li>Shuart, N. G., Haitin, Y., Camp, S. S., Black, K. D., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+for+3:1+subunit+stoichiometry+of+rod+cyclic+nucleotide-gated+ion+channels.">Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels.</a> Nat Commun. 2, 457 (2011).</li><li>Chang, S. Y., Tsai, P. C., Tseng, C. S., Liang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refolding+and+characterization+of+a+yeast+dehydrodolichyl+diphosphate+synthase+overexpressed+in+Escherichia+coli.">Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.</a> Protein Expr Purif. 23 (3), 432-439 (2001).</li><li>Chang, S. Y., Ko, T. P., Liang, P. H., Wang, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+mechanism+revealed+by+the+crystal+structure+of+undecaprenyl+pyrophosphate+synthase+in+complex+with+sulfate,+magnesium,+and+triton.">Catalytic mechanism revealed by the crystal structure of undecaprenyl pyrophosphate synthase in complex with sulfate, magnesium, and triton.</a> J Biol Chem. 278 (31), 29298-29307 (2003).</li><li>Guo, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+undecaprenyl+pyrophosphate+synthase+in+complex+with+magnesium,+isopentenyl+pyrophosphate,+and+farnesyl+thiopyrophosphate:+roles+of+the+metal+ion+and+conserved+residues+in+catalysis.">Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis.</a> J Biol Chem. 280 (21), 20762-20774 (2005).</li><li>Collins, F. S., Green, E. D., Guttmacher, A. E., Guyer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vision+for+the+future+of+genomics+research.">A vision for the future of genomics research.</a> Nature. 422 (6934), 835-847 (2003).</li><li>Freeze, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+human+glycosylation+disorders:+biochemistry+leads+the+charge.">Understanding human glycosylation disorders: biochemistry leads the charge.</a> J Biol Chem. 288 (10), 6936-6945 (2013).</li><li>LaVallie, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thioredoxin+gene+fusion+expression+system+that+circumvents+inclusion+body+formation+in+the+E.+coli+cytoplasm.">A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.</a> Biotechnology (N Y). 11 (2), 187-193 (1993).</li><li>Elena, C., Ravasi, P., Castelli, M. E., Peiru, S., Menzella, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+codon+optimized+genes+in+microbial+systems:+current+industrial+applications+and+perspectives.">Expression of codon optimized genes in microbial systems: current industrial applications and perspectives.</a> Front Microbiol. 5, 21 (2014).</li><li>Vincentelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+protein+expression+screening+and+purification+in+Escherichia+coli.">High-throughput protein expression screening and purification in Escherichia coli.</a> Methods. 55 (1), 65-72 (2011).</li></ol>

Protokollen beskrevet her for rensning af funktionelle humane DHDDS i E. coli- celler er simpel og effektiv, hvilket tillader at man overudtrykker og renser proteinet i 3 - 4 dage, når en egnet konstruktion er tilgængelig. Sådanne protokoller til proteinrensning er af særlig betydning givet gennembrudene i genom-sekventering, hvilket tilvejebragte overflod af information vedrørende genetik hos mange sygdomme 18 , hvorved der kræves udvikling af høj-gennemstrømningsmetoder til kar...

Prenyltransferaser er en gruppe enzymer, der katalyserer kædeforlængelse af allylldifosfat under anvendelse af isopentenyldiphosphat (IPP) via multiple kondensationsreaktioner 1 , 2 . Z-type enzymer katalyserer dannelsen af cis- dobbeltbindinger fra kondensationsreaktionen, hvorimod E-type enzymer katalyserer trans- dobbeltbindingsdannelse 3 . cis -Prenyltransferases (cis -PT, Z-type enzymer) er klassisk klassificeres efter deres produkt kædelængde i kortkædede (C 15), medium-kæde (C 50-55), og langkædede (C 70-120 ) 4 . DHDDS (dehydrodolichyldiphosphatsyntase) er en eukaryotisk langkædede cis -PT, der katalyserer kædekonstruktion af farnesyldiphosphat (FPP, et allylldifosfat) via multiple kondensationer med isopentenyldiphosphat (IPP) 15 , 6 . Dette resulterer i dannelsen af ​​dehydrodolichyldiphosphat, et C 55-100 polyprenyldiphosphat, der tjener som en precursor for dolichylpyrophosphat, glycosylbærermolekylet involveret i N-koblet proteinglycosylering 1 . Blandt Ashkenazi-jøder resulterer en missens ikke-konservativ mutation (K42E) i DHDDS i autosomal recessiv retinitis pigmentosa 7 , 8 . Derfor blev den nuværende protokol udviklet med henblik på at erhverve renset DHDDS egnet til mekanistiske undersøgelser. Escherichia coli betragtes som den mest hensigtsmæssige og omkostningseffektive vært til rekombinant proteinekspression og er derfor også den hyppigst anvendte vært. Men når man forsøger at heterologt overudtrykke proteiner i E. coli , bør der laves proteinspecifikke overvejelser. Fås korrekt foldet, aktivE rekombinante proteiner fra E. coli , er ikke et simpelt spørgsmål på grund af de forskellige egenskaber af forskellige proteiner. Talrige tilgange er blevet udviklet for at overvinde disse forhindringer. Her blev brugen af ​​proteinfusion, optimerede kulturbetingelser og codonoptimering anvendt til at tillade overekspression og oprensning af funktionelt aktive humane DHDDS i E. coli . Fremhæves, en tidligere forsøg på at overudtrykke gær cis -PT uden proteinfusion mislykkedes på grund af fuldstændig uopløselighed selv i nærvær af detergent 12. Den beskrevne protokol er enkel, omkostningseffektiv, tidsbesparende og giver en mulighed for at opnå DHDDS-præparater, der er egnede til mekanistiske undersøgelser. I betragtning af homologien af cis -PT blandt forskellige arter foreslår vi, at denne protokol også kan anvendes til anden eukaryot cis -PT.

<table width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

Prenyltransferaser (PT) er en gruppe enzymer, som katalyserer kædelængelse af allylicdiphosphat under anvendelse af isopentenyldiphosphat (IPP) via multiple kondensationsreaktioner. DHDDS (dehydrodolichyldiphosphatsyntase) er en eukaryotisk langkædede cis -PT (dannende cis- dobbeltbindinger fra kondensationsreaktionen), som katalyserer kædekonstruktion af farnesyldiphosphat (FPP, et allylicdiphosphat) via multiple kondensationer med isopentenyldiphosphat (IPP). DHDDS er af biomedicinsk betydning, da en ikke-konservativ mutation (K42E) i enzymet resulterer i retinitis pigmentosa , hvilket i sidste ende fører til blindhed. Derfor blev den nuværende protokol udviklet med henblik på at erhverve store mængder renset DHDDS, der er egnet til mekanistiske undersøgelser. Her blev brugen af ​​proteinfusion, optimerede kulturbetingelser og codonoptimering anvendt til at tillade overekspression og oprensning af funktionelt aktive humane DHDDS i <em&gt; E. Coli. Den beskrevne protokol er enkel, omkostningseffektiv og tidsbesparende. Homologien af cis -PT blandt forskellige arter antyder, at denne protokol kan anvendes til andre eukaryotiske cis -PT såvel som dem, der er involveret i syntetisk gummisyntese.

Generel oversigt over konstruktionen anvendt her og rensningsprocessen er vist i figur 1 . Prøverne opnået ved hvert oprensningstrin er vist i figur 2 . Denne SDS-PAGE analyse viser trinvis oprensning af DHDDS, hvilket resulterer i et stærkt oprenset produkt. Figur 3 viser resultaterne af analytisk SEC af det oprensede enzym, hvilket afslører, at proteinet kun observeres som en homodimer. <stron...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

En simpel protokol til overekspression og oprensning af codonoptimeret human cis- phenyltransferase under ikke-denaturerende betingelser fra Escherichia Coli , er beskrevet sammen med et enzymatisk aktivitetsassay. Denne protokol kan generaliseres til fremstilling af andre cis- prenyltransferase proteiner i mængde og kvalitet egnet til mekanistiske undersøgelser.

Overekspression og oprensning af mennesket<em&gt; Cis</em&gt; -prenyltransferase i<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Research

JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

Vurdering af Somatisk Hypermutation i Ramos B-celler efter Overekspression eller Knockdown af specifikke gener

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Ændring af primære humane T-celler

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Non-invasive Model of Neuropathogenic<em&gt; Escherichia coli</em&gt; Infektion i Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Et-trins Negativ kromatografisk oprensning af<em&gt; Helicobacter pylori</em&gt; Neutrofilaktiverende protein overudtrykt i<em&gt; Escherichia coli</em&gt; I Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Flowcytometrisk analyse of Natural Killer Cell lytisk aktivitet i humant fuldblod

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Påvisning af Enterohemorrhagic Escherichia Coli kolonisering i Murine vært ved Non-invasiv In Vivo bioluminescens System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Oprettelsen af en tætte Transposon indsættelse bibliotek ved hjælp af bakteriel konjugation i Enterobacterial stammer som Escherichia Coli eller Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

En letkøbt protokol til at generere Site-Specifically acetyleret proteiner i Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...