Name: identifying amino acid overproducers using rare-codon-rich markers
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काम संयुक्त रूप से चीन के राष्ट्रीय प्राकृतिक विज्ञान फाउंडेशन द्वारा समर्थित किया गया था (नहीं. 21676026), चीन के राष्ट्रीय कुंजी अनुसंधान एवं विकास कार्यक्रम (अनुदान संख्या 2017YFD0201400), और चीन Postdoctoral विज्ञान फाउंडेशन (नहीं. 2017M620643). UCLA उन्नति संस्थान (Suzhou) में काम करता है Jiangsu प्रांत और सूज़ौ औद्योगिक पार्क से आंतरिक अनुदान द्वारा समर्थित थे.

1. दुर्लभ-कोडोन समृद्ध मार्कर जीनों को व्यक्त करने वाले प्लाज्मिड का निर्माण
<ol><li>एक मार्कर जीन है कि लक्षित एमिनो एसिड के लिए आम codons की एक उचित संख्या में शामिल का चयन करें. नोट: एल-ल्यूसिन के लिए, कानामाइ?...

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मार्कर जीन और चयन या स्क्रीनिंग माध्यम में दुर्लभ codons की संख्या दुर्लभ codon संशोधित मार्कर जीन से प्रोटीन अभिव्यक्ति को बाधित करने के लिए महत्वपूर्ण हैं. यदि कोई महत्वपूर्ण अंतर जंगली प्रकार मार्कर जीन ?...

एमिनो एसिड की वर्तमान उत्पादन किण्वन पर काफी निर्भर करता है. हालांकि, सबसे एमिनो एसिड उत्पादन उपभेदों के लिए titers और पैदावार वैश्विक एमिनो एसिड बाजार है कि अरबों डॉलर1,2के लायक है की बढ़ती मांग से नीचे हैं. उच्च प्रदर्शन एमिनो एसिड overproducers प्राप्त एमिनो एसिड उद्योग के उन्नयन के लिए महत्वपूर्ण हैं.
एमिनो एसिड ओवरप्रोड्यूसर की पहचान करने की पारंपरिक रणनीति एमिनो एसिड और प्रोटीन संश्लेषण3,4में उनके एनालॉग के बीच की प्रतियोगिताओं का फायदा उठाती है . इन एनालॉग tRNAs कि इसी अमीनो एसिड पहचान और इस तरह पेप्टाइड चेन की दीर्घों को बाधित चार्ज करने में सक्षम हैं, गिरफ्तार विकास या सेल मौत5के लिए अग्रणी . एनालॉग तनाव का विरोध करने के लिए एक तरीका intracellular एमिनो एसिड की सांद्रता में वृद्धि करने के लिए है. समृद्ध एमिनो एसिड सीमित tRNAs के लिए एनालॉग बाहर प्रतिस्पर्धा और कार्यात्मक प्रोटीन का सही संश्लेषण सुनिश्चित करेगा. इसलिए, उपभेदों कि एनालॉग जीवित चयनित किया जा सकता है और संभावना इसी अमीनो एसिड के overproducers हैं.
हालांकि इस तरह के एल-ल्यूसिन6के रूप में एमिनो एसिड के लिए overproducers के चयन में सफल साबित कर दिया, अनुरूप आधारित रणनीति गंभीर कमियों से ग्रस्त है. एक प्रमुख चिंता का विषय अनुरूप प्रतिरोध mutagenesis की प्रक्रिया से या सहज उत्परिवर्तनों के माध्यम से उत्पन्न है. प्रतिरोध के साथ तनाव अवरुद्ध, निर्यात, या एनालॉग5अपमानजनक द्वारा चयन से बच सकते हैं। एक अन्य चिंता का विषय अन्य सेलुलर प्रक्रियाओं7पर एनालॉग के विषाक्त दुष्प्रभाव है. एक परिणाम के रूप में, उपभेदों कि एनालॉग चयन जीवित अमीनो एसिड overproducers नहीं हो सकता है, जबकि वांछित overproducers झूठा नकारात्मक पक्ष प्रभाव के कारण समाप्त किया जा सकता है.
यहाँ, codon पूर्वाग्रह के कानून पर आधारित एक उपन्यास रणनीति के क्रम में एमिनो एसिड overproducers की सही और तेजी से पहचान प्राप्त करने के लिए प्रस्तुत किया है. अधिकांश अमीनो अम्लों को एक से अधिक न्यूक्लिओटाइड त्रिक द्वारा एनकोडेड किया जाता है जो मेजबान जीवों8,9द्वारा अलग ढंग से अनुकूल होता है . कुछ codons शायद ही कभी कोडन दृश्यों में उपयोग किया जाता है और दुर्लभ codons के रूप में जाना जाता है. एमिनो एसिड में उनके अनुवाद cognate tRNAs कि इसी अमीनो एसिड ले पर भरोसा करते हैं. हालांकि, tRNAs कि दुर्लभ codons पहचान आम तौर पर आम codons10,11के tRNAs की तुलना में बहुत कम बहुतायत है. नतीजतन, इन दुर्लभ tRNAs कम अन्य isoacceptors के साथ प्रतियोगिताओं में मुक्त एमिनो एसिड पर कब्जा करने की संभावना है, और दुर्लभ codon अमीर दृश्यों के अनुवाद में कमी या यहां तक कि समाप्त कर रहे हैं जब एमिनो एसिड की मात्रा सीमित हैं शुरू 10. अनुवाद, सैद्धांतिक रूप से बहाल किया जा सकता है अगर वहाँ एक एमिनो एसिड अधिशेष है के बाद पर्याय आम tRNAs चार्ज करने के बाद overproductions या इसी एमिनो एसिड12के अतिरिक्त feedings . यदि दुर्लभ-कोडोन समृद्ध जीन एक चयन या स्क्रीनिंग मार्कर encodes, इसी phenotypes का प्रदर्शन उपभेदों तो आसानी से पहचान की जा सकती है और संभावना लक्षित एमिनो एसिड के overproducers हैं.
उपरोक्त रणनीति एक चयन और एमिनो एसिड overproducers की पहचान के लिए एक स्क्रीनिंग प्रणाली स्थापित करने के लिए लागू किया जाता है. चयन प्रणाली एंटीबायोटिक प्रतिरोध जीन (जैसे, केएनआर) मार्कर के रूप में उपयोग करता है, जबकि स्क्रीनिंग सिस्टम फ्लोरोसेंट एन्कोडिंग जीन का उपयोग करता है (जैसे, ग्रीन फ्लोरोसेंट प्रोटीन [GFP]) या क्रोमोजेनिक (जैसे, PrancerPurple) प्रोटीन। दोनों प्रणालियों में मार्कर जीन अपने पर्यायन दुर्लभ विकल्प के साथ लक्षित एमिनो एसिड के लिए आम codons की परिभाषित संख्या की जगह द्वारा संशोधित कर रहे हैं. उत्परिवर्तन पुस्तकालय है कि दुर्लभ codon अमीर मार्कर जीन बंदरगाह में तनाव का चयन कर रहे हैं या उचित परिस्थितियों में जांच की, और लक्षित एमिनो एसिड के overproducers आसानी से पहचान की जा सकती है. कार्यप्रवाह दुर्लभ-कोडोन समृद्ध मार्कर जीन प्रणाली के निर्माण के साथ शुरू होता है, जिसके बाद काम की स्थिति के अनुकूलन, और फिर एमिनो एसिड overproducers की पहचान और सत्यापन. इस अनुरूप स्वतंत्र रणनीति प्रोटीन अनुवाद में हठधर्मिता पर आधारित है और व्यावहारिक रूप से एमिनो एसिड overproducers की सटीक और तेजी से पहचान सक्षम करने के लिए सत्यापित किया गया है. सैद्धांतिक रूप से, यह सीधे दुर्लभ codons के साथ और सभी सूक्ष्मजीवों के लिए एमिनो एसिड के लिए नियोजित किया जा सकता है. सभी में, दुर्लभ codon आधारित रणनीति पारंपरिक अनुरूप आधारित दृष्टिकोण के लिए एक कुशल विकल्प के रूप में काम करेंगे जब विशिष्ट एमिनो एसिड के लिए उचित एनालॉग उपलब्ध नहीं हैं, या जब एक उच्च झूठी सकारात्मक दर प्रमुख चिंता का विषय है. नीचे दिए गए प्रोटोकॉल में ल्यूकोइन दुर्लभ कोडोन का उपयोग करता है ताकि Escherichia कोलाई एल-ल्यूसिन ओवरप्रोड्यूसर की पहचान करने में इस रणनीति को प्रदर्शित किया जा सके।

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo</td>
			<td>51101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure HiPure Plasmid MiniPrep Kit</td>
			<td>Transgen</td>
			<td>EM111-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>Transgen</td>
			<td>EG101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson assembly master mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane</td>
			<td>Thermo</td>
			<td>H3061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenyl isothiocyanate</td>
			<td>Sigma</td>
			<td>P1034</td>
			<td></td>
		</tr>
		<tr>
			<td>PrancerPurple CPB-37-441</td>
			<td>ATUM</td>
			<td>CPB-37-441</td>
			<td></td>
		</tr>
		<tr>
			<td>TransStar FastPfu Fly DNA polymerase</td>
			<td>Transgen</td>
			<td>AP231-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-high performance liquid chromatography</td>
			<td>Agilent</td>
			<td>1290 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild type C. glutamicum</td>
			<td>ATCC</td>
			<td>13032</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold E. coli competent cell</td>
			<td>Agilent</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX RRHD Eclipse Plus C18 column</td>
			<td>Agilent</td>
			<td>959759-902K</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying amino acid overproducers using rare-codon-rich markers

एमिनो एसिड के लिए बढ़ती बाजार को संतुष्ट करने के लिए, उच्च प्रदर्शन उत्पादन उपभेदों की जरूरत है. एमिनो एसिड overproducers पारंपरिक एमिनो एसिड और उनके एनालॉग के बीच प्रतियोगिताओं का दोहन द्वारा पहचान कर रहे हैं. हालांकि, इस एनालॉग आधारित विधि कम सटीकता की है, और विशिष्ट अमीनो एसिड के लिए उचित एनालॉग सीमित हैं. यहाँ, हम एक वैकल्पिक रणनीति है कि दुर्लभ codon अमीर मार्करों का उपयोग एमिनो एसिड overproducers के एक सटीक, संवेदनशील, और उच्च throughput स्क्रीनिंग सक्षम बनाता है प्रस्तुत करते हैं. इस रणनीति प्रोटीन अनुवाद में codon उपयोग पूर्वाग्रह की घटना से प्रेरित है, जिसके लिए codons कोडिंग डीएनए में घटना की उनकी आवृत्तियों के आधार पर आम या दुर्लभ लोगों में वर्गीकृत कर रहे हैं. दुर्लभ codons का अनुवाद उनके इसी दुर्लभ हस्तांतरण RNAs (tRNAs), जो पूरी तरह से भुखमरी के तहत cognate अमीनो एसिड द्वारा चार्ज नहीं किया जा सकता पर निर्भर करता है. सैद्धांतिक रूप से, दुर्लभ tRNAs चार्ज किया जा सकता है अगर वहाँ पर्याय आम समग्राही चार्ज करने के बाद एमिनो एसिड का अधिशेष है. इसलिए, दुर्लभ codons की वजह से मंद अनुवाद खिला या इसी एमिनो एसिड के intracellular overproductions द्वारा बहाल किया जा सकता है. इस धारणा के तहत, एमिनो एसिड overproducers की पहचान करने के लिए एक चयन या स्क्रीनिंग प्रणाली एंटीबायोटिक प्रतिरोध जीन या जीन में अपने पर्याय का दुर्लभ विकल्प के साथ लक्षित एमिनो एसिड के आम codons की जगह द्वारा स्थापित किया गया है एन्कोडिंग फ्लोरोसेंट या क्रोमोजेनिक प्रोटीन. हम बताते हैं कि प्रोटीन अभिव्यक्ति बहुत दुर्लभ codons के समावेश द्वारा बाधित किया जा सकता है और प्रोटीन के स्तर एमिनो एसिड सांद्रता के साथ सकारात्मक सहसंबंधित. इस प्रणाली का उपयोग करना, कई एमिनो एसिड के overproducers आसानी से उत्परिवर्तन पुस्तकालयों से बाहर जांच की जा सकती है. इस दुर्लभ-कोडोन आधारित रणनीति केवल एक संशोधित जीन की आवश्यकता है, और मेजबान कम अन्य तरीकों की तुलना में चयन से बचने की संभावना है. यह एमिनो एसिड overproducers प्राप्त करने के लिए एक वैकल्पिक दृष्टिकोण प्रदान करता है.

चयन प्रणाली के लिए, दुर्लभ codon अमीर एंटीबायोटिक प्रतिरोध जीन शरण उपभेदों के लिए OD600 में एक तेज कमी जंगली प्रकार एंटीबायोटिक प्रतिरोध जीन शरण तनाव की तुलना में मनाया जाना चाहिए जब एक उपयु?...

Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

इस अध्ययन के दुर्लभ codon अमीर मार्करों का उपयोग करके एमिनो एसिड overproducers की पहचान करने में पारंपरिक विषाक्त अनुरूप आधारित विधि के लिए एक वैकल्पिक रणनीति प्रस्तुत करता है सटीकता को प्राप्त करने के लिए, संवेदनशीलता, और उच्च throughput एक साथ.

दुर्लभ-कोडोन-रिच मार्करों का उपयोग कर एमिनो एसिड ओवरप्रोड्यूसर की पहचान करना

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

identifying-amino-acid-overproducers-using-rare-codon-rich-markers

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

HCN1 और TRIP8b बीच प्रोटीन, प्रोटीन बातचीत के छोटे अणु inhibitors की पहचान के लिए विधि

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

वोल्टेज क्लैंप फ्लोराइट्री में<em&gt; Xenopus</em&gt; फ्लोरोसेंट अस्वास्थ्यकर एमिनो एसिड का प्रयोग करने वाले ओक्साइट्स

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

एक नी-शिफ़-बेस की हाइड्रोलिसिस परिसर का उपयोग स्थितियां एसिड अस्थायी रक्षा समूह की अवधारण के लिए उपयुक्त

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

न्यूक्लिक एसिड जांच का उपयोग कर न्यूक्लीज गतिविधि का काइनेटिक स्क्रीनिंग

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

संभावित एपिजेनेटिक मार्कर के टॉप डाउन मास स्पेक्ट्रोमेट्री प्रोफाइलिंग के लिए ज्वार लीफ टिश्यू से हिस्टोन का अलगाव

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

थिओग्लिकोलिक एसिड (टीजीए) का उपयोग करके पौधे बायोमास लिग्निन सामग्री का अनुमान

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

फैटी Acylated प्रोटीन का पता लगाने के लिए Alkynyl फैटी एसिड Analogs के अनुकूलित निगमन क्लिक रसायन विज्ञान का उपयोग कर

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Folding and Probing of Nucleic Acid Constructs

Begin by pipetting G4-TBA, single-stranded TBA, and double-stranded TBA solutions into three different tubes for folding. Heat the samples to 95 degrees Celsius for five minutes. Let them cool slowly to room temperature.Once the tubes have cooled to room temperature, centrifuge the tubes. Next, organize three sets each of seven empty autoclaved 0.5-milliliter tubes. Add three microliters of ultrapure water into each tube.Pipette five microliters of the folded nucleic acid constructs into each tube of the set. Next, add two microliters of either 25 or 250-micromolar B-CEP1 to obtain the final probe concentration of five and 50 micromolar respectively. To the three control tubes, add ultrapure water instead of the compound and incubate all the samples at 37 degrees Celsius.Stop the reaction by placing the tubes at 20 degrees Celsius, then dry the samples in a vacuum centrifuge.

न्यूक्लिक एसिड संरचनाओं की तह और जांच

<meta charset="utf8"></head><body>प्रोटीन-प्रोटीन बाइंडिंग इंटरफेस के अमीनो एसिड वरीयताओं की भविष्यवाणी करना

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

<meta charset="utf8"></head><body>लेखक स्पॉटलाइट: मल्टीस्पेसिफिक प्रोटीन-प्रोटीन इंटरैक्शन में अमीनो एसिड वरीयताओं को समझने के लिए एक कम्प्यूटेशनल दृष्टिकोण

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.