Industrial case study: Evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli

Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.     

Artifacts related to N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide derivatization of citrulline revealed by gas chromatography–mass spectrometry using both electron and chemical ionization

Derivatization with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) was used for gas chromatography–mass spectrometry (GC–MS) analysis of citrulline and ornithine. Aqueous 50 μl aliquots at 1 and 10 mM concentrations were dried and derivatized separately, and 70 eV electron ionization or CH₄ positive chemical ionization were used. Ornithine produced a single GC peak. Physiological citrulline concentrations produced GC artifact peaks for the ornithine derivative, and a compound consistent with elimination of a water molecule from the tri-tert-butyldimethylsilyl (TBDMS) citrulline derivative. A third GC peak obtained using 10 mM citrulline concentrations gave a mass spectrum consistent with a mixture of true tri- and tetra-TBDMS citrulline. Analyses of ¹³C-ureido-labeled citrulline confirmed the presence of the true TBDMS citrulline derivatives produced from 10 mM samples and provided evidence that the TBDMS ornithine artifact results from loss of TBDMS isocyanate from tetra-TBDMS citrulline. Linear-programmed temperature GC retention index data relative to n-alkanes are reported for observed GC peaks.     

Novel Chiral Polyoxometalate: First Arginine Based Anderson-type Complex

A novel chiral polyoxometalate(POM),{[Na(Arg)2]NaH}[AlMo6(OH)6O18].6H2O(1,Arg=L-arginine) has been synthesized by the reaction between Na3[AIMo6(OH)6O18]-8H2O and Arg in aqueous solution and was struct...     

精氨酸的流动注射化学发光法测定

在碱性介质中,精氨酸对硫氰化钾 鲁米诺化学发光体系有增敏作用,建立了用硫氰化钾 鲁米诺 精氨酸体系测定精氨酸的新方法。用该方法测定精氨酸的线性范围为0.01～2.0μg/mL,检出限0.01μg/mL,采样频率为190次/h,对3μg/mL的精氨酸连续平行测量10次,RSD为1.2%。用该方法对皮革屑中酶法提取的精氨酸进行了测定,并与氨基酸测定仪测定的结果进行了比较。     

Effects of l-arginine on aggregates of fatty-acid/potassium soap in the aqueous media

In the present study, we investigated the effects of l-arginine on aggregates of fatty acid/fatty soap in the aqueous media as a function of pH, by means of hydrogen ion titration, viscoelastic measurement, cryo-transmission electron microscopy and phase contrast microscopy. We found out that l-arginine effectively inhibits the oil droplet growth of oleic acid or octanoic acid. The effect is explained in terms of the adsorption of arginine at the microscopic drop surface, or at the oil/water microinterface through the hydrophobic effect assisted by the hydrogen bonds between carboxyl group of fatty acid and carboxylate of arginine. As to the crystallization of lauric acid at temperatures below the melting point of the hydrocarbon chain, arginine is not effective. In addition, we also found out that the strong binding of arginine cation to anionic oleate micells induces the dominant micellar growth. l-arginine has been used in many refolding systems to suppress protein aggregation. These effects of l-arginine on the aggregates of fatty acid/fatty soap in the aqueous media observed in the present study is expected to form a basis to the specific function displayed in the protein refolding.     

Quantum molecular dynamics simulation for fragmentation of arginine molecule induced by ion impact

15 keV Ga⁺ ion impact and the thermal decomposition of arginine molecules has been simulated by quantum molecular dynamics (QMD). We obtained the calculated mass spectra which express the distribution for fragment molecule generated by ion impact or thermal decomposition. From the comparison between the experimental TOF-SIMS spectra and the calculated spectra, we discussed the fragmentation mechanism of arginine molecule by Ga⁺ ion impact.     

Synthesis and biological evaluation of novel phenothiazine derivatives as non-peptide arginine vasopressin V2 receptor antagonists

A series of novel phenothiazine derivatives was synthesized and tested for arginine vasopressin receptor antagonist activity. They were synthesized as novel arginine vasopressin receptor antagonists from phenothiazine as a scaffold via successive acylation, reduction and acylation reactions. Their structures were characterized by 1HNMR, 13CNMRandHRMS, and biological activitywas evaluated by in vitro and in vivo studies. The in vitro binding assay indicated that several compounds are potent selective V2 receptor antagonists. Compounds with promising binding affinity to V2 receptors were selected to conduct the in vivo diuretic studies on Sprague-Dawley rats. Among them, 1n, 1r, 1t and 1v exhibited excellent diuretic activity, especially 1r and 1v. Therefore, 1r and 1v are potent novelAVP V2receptor antagonist candidates.     

An expedient access to chromanols via an arginine-mediated cascade cyclisation in water

A mild and efficient protocol for the synthesis of chromanols has been elaborated via an arginine-mediated cascade cyclisation reaction between salicylaldehyde and methyl vinyl ketone in water. Various substituted salicylaldehydes were successfully used in the aqueous based reaction, affording the chromanol adducts in very good yields. Mechanistically, the reaction presumably proceeds via an oxa-Michael addition followed by an intramolecular aldol addition pathway; the lack of stereoselectivity in the formation of the chromanols alludes to a non-covalent interaction of the amino acid with the substrates as the most plausible interpretation for the activation.     

ATP合酶中α亚基推动γ亚基旋转的可能机制

F1F0-ATP合酶是一种分子马达,它能够利用跨膜的质子浓度梯度推动γ亚基旋转,进而在α/β亚基界面上催化合成ATP,也能够利用ATP水解推动γ亚基旋转,将ATP中蕴藏的化学能转变成为机械能.ATP水解发生在α/β亚基界面上.但是在ATP水解过程中,高能磷酸键断裂释放的能量如何传递给γ亚基,使之旋转?什么原因导致了α/β亚基产生相对运动?本文提出的假说认为:ATP在与α/β界面上的关键氨基酸αArg376、βArg182的相互作用过程中,使Arg的结构由原来的卷曲型转变为伸展型,从而引发催化活性区域的空间构象发生变化,进而推动α/β亚基的相对运动.α/β亚基之间的相对运动传递给γ亚基并使之旋转.     

甘草次酸精氨酸盐的波谱学表征

甘草次酸是甘草的主要活性物质之一,具有抗炎、抗溃疡、抗病毒、防治肿瘤等多种药理活性[1-1]。本文利用精氨酸与甘草次酸成盐,一方面改善了甘草次酸在水中的溶解度,可提高其生物利用度;另一方面可降低血管痉挛的发生[3],从而提高用药安全性。本文主要报道该化合物的波谱学特征。1实验部分1.1甘草次酸精氨酸盐的合成在反应瓶中加入等物质量的18β-甘草次酸(0·95g)和L-精氨酸(0·35g),用80%乙醇75mL溶解,室温下搅拌反应2h,减压除溶剂并干燥,得白色疏松粉末状物,即为甘草次酸精氨酸盐(GA-Arg)。其熔点为184℃~186℃,结构如图1所示。图1甘草…