Pressure induced changes in proteins studied in the diamond anvil cell with Raman spectroscopy

Abstract

The pressure induced denaturation of serum albumin and lysozyme is studied. Preliminary results suggest that the changes induced in serum albumin are reversible below 10 kbar.     

Nanoencapsulation of Fullerenes in Organic Structures with Nonpolar Cavities

The formation of supramolecular structures, assemblies, and arrays held together by weak intermolecular interactions and non-covalent binding mimicking natural processes has been used in applications being anticipated in nanotechnology, biotechnology and the emerging field of nanomedicine. Encapsulation of C₆₀ fullerene by cyclic molecules like cyclodextrins and calixarenes has potential for a number of applications. Similarly, biomolecules like lysozyme also have been shown to encapsulate C₆₀ fullerene. This poster article reports the recent trends and the results obtained in the nanoencapsulation of fullerenes by biomolecules containing nonpolar cavities. Lysozyme was chosen as the model biomolecule and it was observed that there is no covalent bond formed between the bimolecule and the C₆₀ fullerene. This was confirmed from fluorescence energy transfer studies. UV–Vis studies further supported this observation that it is possible to selectively remove the C₆₀ fullerene from the nonpolar cavity. This behavior has potential in biomedical applications     

Partial least square analysis of lysozyme near-infrared spectra

Proteins possess strong absorption features in the combination range (5000-4000 cm⁻¹) of the near infrared (NIR) spectrum. These features can be used for quantitative analysis. Partial least squares (PLS) regression was used to analyze NIR spectra of lysozyme with the leave-one-out, full cross-validation method. A strategy for spectral range optimization with cross-validation PLS calibration was presented. A five-factor PLS model based on the spectral range between 4720 and 4540 cm⁻¹ provided the best calibration model for lysozyme in aqueous solutions. For 47 samples ranging from 0.01 to 10 mg/mL, the root mean square error of prediction was 0.076 mg/mL. This result was compared with values reported in the literature for protein measurements by NIR absorption spectroscopy in human serum and animal cell culture supernatants.     

Quenching interactions and nonexponential decay: tryptophan 138 of bacteriophage T4 lysozyme

Site-directed mutagenesis has been used to prepare variants of bacteriophage T4 lysozyme that contain only one tryptophan residue at position 138 and to change the residues in the immediate environment of this buried residue. Replacement of glutamine-105 by alanine results in a 2.7-fold increase in fluoresence quantum yield and converts the fluorescence decay from a highly nonexponential form to a single-exponential decay. This is atributed to electron transfer quenching of tryptophan-138 fluorescence by glutamine-105. Replacemeent of alanine-146 by threonine results in a 1.6-fold decrease in fluorescence intensity, indicating enhanced quenching by glutamine-105; replacement of glutamine-105 by alanine in this species results in a 5-fold in crease in fluorescence intensity. The interpretation of the nonexponential decay of the glutamine-105-containing species is discussed in terms of reversibility of the quenching process.     

Sequential adsorption of bacteriophage T4 lysozyme stability variants at solid–water interfaces

The sequential adsorption of the wild type T4 lysozyme and one of its structural stability variants was studied, using ellipsometry and ¹²⁵I radioisotope labeling techniques. The mutant lysozyme was produced by substitution of the isoleucine residue at position 3 in the wild type with a tryptophan residue, resulting in a protein with lower structural stability. The mutant protein was more resistant to surfactant-mediated elution, and apparently adsorbed at the interfaces with a greater interfacial area/molecule than the wild typeT4 lysozyme. However, the results of each type of experiment suggested that sequential adsorption and exchange of proteins occurred only in the case of the less stable mutant followed by the wild type. This suggests that, in these exchange reactions, properties of the adsorbing protein (e.g. its ability to adsorb when a relatively small amount of unoccupied area is present) were more important than the apparent binding strength of the adsorbed protein molecules.     

溶菌酶涂膜保鲜蔬菜的研究

选用溶茵酶作为涂膜材料，对黄瓜进行涂膜保鲜，于室温下贮藏，分别测定黄瓜的失重率，Vc含量和叶绿素含量，并通过观察黄瓜在涂膜期间品质的变化，确定最佳涂膜配方，为黄瓜在室温下短期内贮藏及延长其货架期提供一种新方法。     

溶菌酶及其应用

溶菌酶又称胞壁质酶。是一种无毒、无害、安全性很高的高盐基水解蛋白酶,且具有一定的保健作用。作为天然防腐剂的溶菌酶能选择性地分解微生物及植物细胞壁。而对没有细胞壁的人体细胞不会产生降解作用。因而被广泛应用于医药、食品等行业。本文从溶菌酶的组成、结构、性质出发,从食品、医药和科研几个方面详细论述了溶菌酶的应用及其发展前景。     

人溶菌酶N端与Exendin-4嵌合蛋白的基因克隆及原核表达

目的:克隆嵌合多肽人溶菌酶N端-Exendin-4基因并进行原核表达和纯化.方法:通过重组PCR技术将人溶菌酶N端74个氨基酸的基因序列与Exendin-4多肽基因序列相连接,其间引入一段由凝血酶和二肽基肽酶识别位点组成的连接序列.以嵌合基因hLYZ(N74)-Ex4与质粒pET-32a(+)构建原核表达体,转化大肠杆菌BL21(DE3)并诱导表达.表达蛋白经Ni-NTA亲和层析纯化、Western blotting鉴定;透析复性后,以肠激酶切割并回收目的多肽.结果:重组质粒pET-32a/hLYZ(N74)-Ex4构建正确,目的蛋白主要以包涵体形式存在,37℃诱导4h、IPTG浓度为0.6 mmol/L时表达量最高,约占菌体蛋白总量的30%.Western blotting检测显示重组蛋白为单一清晰条带.重组蛋白经肠激酶切割后,回收得到高纯度的嵌合多肽.结论:成功构建hLYZ(N74)-Ex4嵌合基因的原核表达质粒,高效原核表达并获得高纯度目的蛋白.