Design of carrier tRNAs and selection of four-base codons for efficient incorporation of various nonnatural amino acids into proteins in Spodoptera frugiperda 21 (Sf21) insect cell-free translation system

Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system was expanded to include nonnatural amino acids. Orthogonal tRNAs that work as carriers of nonnatural amino acids in the insect system were explored. Four-base codons for assigning the positions of nonnatural amino acids were also selected. Mutated streptavidin mRNAs that contained different four-base codons were prepared and added to the insect cell-free system in the presence of various tRNAs possessing the corresponding four-base anticodons. The tRNAs were chemically aminoacylated with various types of nonnatural amino acids to examine their incorporation efficiencies. Using p-nitrophenylalanine as the nonnatural amino acid and streptavidin as the target protein, tRNA sequences and the types of four-base codons were optimized to maximize the yield of the nonnatural mutant and to minimize production of full-length proteins that do not contain the nonnatural amino acid. Among the tRNA sequences taken from a variety of tRNAs of nonstandard structures, the tRNA derived from Methanosarcina acetivorans tRNA(Pyl) was the most efficient and orthogonal tRNA. Of the CGGN-type four-base codons, CGGA and CGGG were the most efficient ones for assigning the positions of nonnatural amino acids. p-Nitrophenylalanine and 2-naphthylalanine were efficiently incorporated as in the case of Escherichia coli and rabbit reticulocyte cell-free systems. Much less efficient incorporation was observed, however, for other nonnatural amino acids, indicating that the insect system is less tolerant to the structural diversity of amino acids than the E. coli cell-free system.     

Four-base codon-mediated saturation mutagenesis in a cell-free translation system

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.     

Amber codon-mediated expanded saturation mutagenesis of proteins using a cell-free translation system

Saturation mutagenesis of proteins, in which an amino acid at a specific site is substituted with each of the other 19 amino acids, is a powerful method for protein analysis and engineering. However, 19 mutated genes have to be prepared to express all possible amino acid-substituted proteins at one site. We previously reported a four-base codon-mediated saturation mutagenesis method for the expression of all 20 amino acid-substituted proteins from one four-base codon-containing gene using 20 types of chemically aminoacylated tRNAs corresponding to the four-base codon. In this study, an improved method for saturation mutagenesis using an amber codon was developed. By combining the use of Escherichia coli-derived amber suppressor tRNAs and chemically aminoacylated Mycoplasma-derived tRNAs, all 20 mutated proteins were successfully expressed from one amber mutant gene in a cell-free translation system. The use of E. coli-derived amber suppressor tRNAs simplified the preparation of the tRNA reagents required for saturation mutagenesis, and also improved the expression of some of the mutated proteins. The expressed mutant proteins were used to evaluate the effect of the amino acid substitutions on the ligand-binding activity. To further expand the possibilities of saturation mutagenesis, a series of nonnatural amino acids analogous to a naturally occurring amino acid was added to the amino acid repertoire. The expanded saturation mutagenesis was utilized to evaluate the effect of a series of atomic-level side chain substitutions on the protein activity.     

Isolation of functionally active acini from bovine mammary gland.

Conditions to obtain high yields of intact acini from lactating bovine mammary glands and certain structural and functional characteristics of isolated acini were investigated. A two-factor experiment with three collagenase concentrations (100, 150, and 200 mg/100 ml) and incubation periods (40, 60, and 90 min) demonstrated that increases in both factors significantly increased net acini yield. Largest amounts of acini obtained, based on content of deoxyribonucleic acid, were 10.3% of the original tissue. Morphologically, fractions consisted primarily of acini or large cell clumps, and nearly all cells excluded trypan blue. Acini cultured in complete nutrient medium incorporated radioactive leucine into proteins. When acini were incubated in medium without supplemental amino acids, specific activity of synthesized proteins was correlated negatively with incubation time. During pulse labeling with radioactive L-leucine over 16 min, true labeling of acinar proteins occurred after 4 min. Sequential kinetics of pulse-chase labeling demonstrated a response pattern unique to the in vitro acinar system. Acinar protein synthesis was inhibited by cycloheximide and strongly stimulated by by 3',5'-cyclic adenosine monophosphate.     

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.     

大叶紫薇果仁蛋白质和氨基酸的分析研究

对大叶紫薇果仁蛋白质与氨基酸进行了研究,测得果仁粗蛋白的含量为10.2 % ,其蛋白质组成为:清蛋白3.31%、球蛋白1.43%、醇溶蛋白2.09%、谷蛋白1.54%;含有18 种氨基酸,其中有营养必需的8 种氨基酸. 并根据模式蛋白质,应用化学分析法对果仁蛋白质营养价值进行了评价.     

萝卜籽粕蛋白质的组成及功能性质

以萝卜籽粕为原料,提取了其中的蛋白质;根据溶解性,萝卜籽粕蛋白中的清、球、谷、醇溶蛋白被分 离;用氨基酸自动分析仪测定了萝卜籽粕蛋白的氨基酸组成;物理化学方法测定萝卜籽粕蛋白的功能性质以及体外 清除1,1-二苯基-2-三硝基苯肼自由基、?OH、NO2 －、H2O2的能力。结果表明：萝卜籽粕蛋白中球、谷、清和醇溶蛋 白含量分别占总蛋白质的44%、33%、21%和2%;萝卜籽粕蛋白含18 种氨基酸,第一限制氨基酸为蛋氨酸,其氨基 酸评分为57。萝卜籽粕蛋白具有很好的功能性质及抗氧化能力,表明有很高的开发价值。     

Effects of insulin and amino acids on milk protein concentration and yield from dairy cows.

Our study investigated the effect of insulin on the regulation of milk protein synthesis in well-fed cows (n = 4) with or without additional amino acids (AA). The design was a two-way crossed factorial with two 12-d periods involving abomasal infusions of either water or a mixture of casein (500 g/d) plus branched-chain AA (88 g/d). During the last 4 d of each period a hyperinsulinemic-euglycemic clamp was performed; insulin was infused at 1.0 microgram.kg of BW-1.h-1 to increase circulating levels fourfold, and euglycemia was maintained by infusion of glucose. Cows were fed a diet formulated to exceed requirements for metabolizable energy and protein. During abomasal water infusion, the insulin clamp increased milk protein yields by 15% (+128 g/d); when combined with abomasal infusion of casein plus branched-chain AA, milk protein yield was increased by 25% (+213 g/d). These increases resulted from equivalent increases in milk protein concentration and milk yield. Concentrations of casein and whey proteins in milk were increased by insulin clamp treatments; however, there were no major changes in the relative proportions of individual casein and whey proteins. Plasma concentrations of essential AA were reduced (-33%) during the insulin clamp treatments; effects were most dramatic for the branched-chain AA (-41%) and their keto acids (-45%). Results confirm the important regulatory role of the endocrine system in milk protein synthesis and demonstrate this potential to produce milk protein is not fully expressed.     

Evaluation of mustard (Brassica juncea) protein isolate prepared by steam injection heating for reduction of antinutritional factors

A process for the preparation of mustard protein isolate, comprising steps such as dispersion of defatted meal in 0.1 mol/l NaCl solution, incubation, extraction at alkaline pH, followed by treatment of the protein solution with activated carbon was developed. The protein, coagulated by steam injection, was subjected to separation by centrifugation, washing and spray drying. The parameters evaluated were protein yield, purity, presence of antinutritional factors and nutritional quality of proteins. The protein yield was 58-60%. The purity of the protein isolate was 95%. The hydrolysed products of glocosinolates like isothiocyanates and oxazolidine thione levels, phenolics and phytic acid levels were low in the protein isolate. The in vitro digestibility of the protein isolate was 92.4% compared to 80.6% of the meal. Chemical score of the meal and protein isolate were similar; isoleucine was the first limiting amino acid. The calculated nutritional indices, essential amino acid index, biological value, nutritional index and C-PER of protein isolate were higher compared to meal. The protein quality as indicated by amino acid profile and PDCAAS scores for 10-12-years old and adults were 100.     

改造乙酰羟酸合成酶提高L-亮氨酸产量

乙酰羟酸合成酶（acetohydroxy acid synthase,AHAS,编码基因ilvBN）是L-亮氨酸合成途径的第一个限速酶。以谷氨酸棒杆菌XL-3（Corynebacterium glutamicum XL-3）为底盘细胞,通过分析并改造AHAS增加其对底物丙酮酸的偏好性,从而提高L--亮氨酸产量。首先利用AHAS的氨基酸序列进行同源建模,根据蛋白质结构进行丙氨酸扫描,找到突变的潜在位点,通过测定突变体酶活力和重组菌株的L--亮氨酸产量寻找最适突变体。测定结果发现将157位Gln突变成Arg能够有效提高AHAS催化丙酮酸的能力,最终重组菌株的L--亮氨酸产量达到（23.5±1.8）g/L,比出发菌株谷氨酸棒杆菌XL-3增加了51%,同时副产物L--异亮氨酸产量有所下降。因此,通过对AHAS的理性改造促进了L--亮氨酸的合成,该研究结果对后续利用蛋白质工程强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

Specific N-terminal biotinylation of a protein in vitro by a chemically modified tRNA(fmet) can support the native activity of the translated protein

Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10-20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA(fmet) aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.     

蚕蛹蛋白和蚕蛹短肽的模拟胃肠道消化特性研究

研究了蚕蛹蛋白和蚕蛹短肽的氨基酸组成,并分析了其在模拟胃肠道消化过程中的消化特性和氮释放量,探究蚕蛹蛋白和短肽在人类营养支持方面的应用。使用自动氨基酸分析仪测定蚕蛹蛋白和蚕蛹短肽的氨基酸含量,并计算氨基酸评分(AAS)、化学评分(CS)和必需氨基酸指数(EAAI)。蚕蛹蛋白和蚕蛹短肽的消化特性和氮释放量通过体外模拟胃肠道消化实验和凯氏定氮法进行评价的计算。结果表明,蚕蛹蛋白和蚕蛹短肽含有人体所需的18种氨基酸,而且必需氨基酸的组成合理。蚕蛹蛋白和蚕蛹短肽在体外模拟胃肠道消化实验中有良好高效的消化率和较高的氮释放量(约90%)。因此在今后将蚕蛹蛋白和蚕蛹短肽作为优质且经济的蛋白质原料,在动物饲料和人类肠内营养支持制剂方面有巨大的应用潜力。      

北太平洋鱿鱼(Todarodes pacificus)内脏自溶液总氨基酸组成质量评价和体外抗氧化性分析

采用自溶法水解鱿鱼内脏,测定鱿鱼内脏自溶液(squid viscera autolysates,SVAs)的蛋白质提取率、可溶性氮含量、游离氨基酸含量和水解度,分析SVAs总氨基酸组成,评价其营养价值,并对SVAs的体外抗氧能力进行分析。结果表明:SVAs的蛋白质提取率为(53.89±1.17)%、可溶性氮质量分数为(78.47±1.16)%、游离氨基酸质量浓度为(0.22±0.03)mg/m L、水解度为(14.73±2.02)%。SVAs中必需氨基酸含量占氨基酸总量的37.03%,呈味氨基酸(天冬氨酸、谷氨酸、丙氨酸和甘氨酸)含量占氨基酸总量的42.40%。氨基酸组成与联合国粮农组织/世界卫生组织推荐氨基酸评分标准模式和全鸡蛋蛋白质氨基酸模式相比较,得出SVAs是一种营养丰富、且氨基酸组成合理的优质蛋白源。研究还表明,SVAs具有良好的体外抗氧化能力,其中清除1,1-二苯基-2-三硝基苯肼自由基和羟自由基的IC50值分别为0.24 mg/m L和0.74 mg/m L,还原能力强于相同质量浓度的L-肌肽。     

Structure-function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1-SLT2 pathway in Saccharomyces cerevisiae

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1-dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in-depth structure-function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N- and C-termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80-340) that is able to restore wild-type phenotypes of knr4Delta mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1-dependent pathway. For these crucial events to occur, the presence of the N-terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1-Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries.     

Problem of the rational classification of dietary proteins

A classification of food proteins has been proposed based on two new qualitative parameters: potential biological value (BVp) and compensation coefficient (C). BVp determines the balance degree and agreement with the body requirement of food protein amino acids. The C coefficient estimates the value of the food protein aminogram improvement at the expense of the endogenous essential amino acid reserve. According to these parameters food proteins can be divided into 4 classes. Class I includes proper alimentary specific food proteins (milk and egg proteins) possessing mean values of BVp and high C coefficients. Class II proteins are characterized by rather high BVp values, i.e. by a good balance of essential amino acids and C coefficient reaching 0, they are represented by animal proteins (those of meat and fish), and vegetable proteins (those of soybean, rape and cotton). Class III contains proteins of food grains characterized by low BVp values and C coefficients. Class IV contains proteins with zero values of BVp, containing no essential amino acids (for example, gelatin, hemoglobin, zein), but showing high C coefficients. In such cases the nature of compensation differs from that of class I proteins, it is paradoxic and temporary.     

母乳和不同婴儿配方乳粉中蛋白质消化性研究

以母乳为对照,模拟婴儿胃肠消化环境对两种普通婴儿配方乳粉和一种适度水解蛋白婴儿配方乳粉中的蛋白质进行体外模拟消化研究,测定其体外胃、肠以及胃肠总消化率和消化液中的氨基酸含量。结果表明体外胃消化率、肠消化率及胃肠总消化率从高到低依次均为:母乳>适度水解蛋白婴儿配方乳粉>婴儿配方乳粉B>婴儿配方乳粉A,且母乳中蛋白质的体外消化率均显著高于适度水解蛋白婴儿配方乳粉和两种普通婴儿配方乳粉(p<0.05)。此外,在体外胃肠总消化液中,适度水解蛋白婴儿配方乳粉中的必需氨基酸总量显著高于母乳和婴儿配方乳粉A、B(p<0.05),母乳和适度水解蛋白婴儿配方乳粉中的氨基酸总量显著高于婴儿配方乳粉A、B(p<0.05)。与两种普通婴儿配方乳粉相比,适度水解蛋白婴儿配方乳粉中蛋白质能更好的被机体消化利用,营养价值更高。      

Effects of barley flour and barley protein isolate on chemical, functional, nutritional and biological properties of Pita bread

This study was carried out to investigate the effects of fortification of wheat flour with barley flour (BF) and barley protein isolate (BPI) at three levels; 5, 10 and 15% levels on the chemical composition, nutritional evaluation and biological properties of pita bread. Proteins fractions such as globulin, prolamin, glutelin-1 and glutelin-2 as well as protein isolates were extracted from barley flour and evaluated for protein yield, chemical composition and nutritional quality. Highest yield and essential amino acids contents were obtained in barley protein isolate. SDS-PAGE gels electrophoresis indicated that fortified wheat flour with BPI and BF consists of proteins coming from wheat flour and barley proteins. The contents of essential limiting amino acids in bread were increased from 1.38 to 3.10 g/100 g for lysine and from 0.86 to 1.73 g/100 g for methionine as the ratio of fortification with BF and BPI increased from 0 to 15%. The highest content of total phenolics, antioxidant activity, and inhibitory activity for both angiotensin converting enzyme (ACE) and α-amylase were found in fortified bread with BPI at 15%. Results indicated that bread made from fortification of wheat flour with BF and BPI at 15% showed superior chemical, physico-chemical, nutritional and biological properties.     

Nucleic acid and protein metabolism in undernutrition and protein deficiency

This review discusses the metabolism of nucleic acids and proteins in various models of undernutrition in female rats and their neonatal and 21-day-old progeny. Based on the observations noted in our laboratories and those of other investigators, it is concluded that body and organ weights as well as various parameters of cellular growth (DNA, RNA, proteins, amino acids and total nucleotides) fail to increase normally in dietary-insulted animals. Protein and RNA synthesis demonstrate variable responses, leading to the speculation that modulation of mRNA metabolism and of protein synthesis occurs in dietary-restricted rats. These findings are also confirmed by the organ weight to DNA ratios. It is further noted that, despite the increases in protein and RNA synthesis in certain organs, protein and RNA register below-normal values, indicating that their degradation is much faster than their formation. This postulate is supported: by the enhanced activities of acid cathepsin (a protein-degrading enzyme) and of RNAse A (a RNA-degrading enzyme); by the elevated concentrations of circulating amino acids and total nucleotides; as well as by the accelerated excretion of nitrogenous compounds in the urine and feces of dietary-restricted animals. Modifications of RNA turnover are also evident in the tRNA and soluble RNA fractions of the liver of dietary-insulted rats. Studies on brain mRNA translatability have revealed: that food deprivation elicits a shorter species of pre-mRNA via a reduced polynucleotide elongation rate; that not all poly A+ RNA sequences present in control rats occur in dietary-restricted animals; and that the translatability of polymerase II is far lower in dietary-insulted rats. Other investigations on the translatability of liver, brain, kidney, spleen and thymus mRNA have demonstrated changes in mRNA via altered protein synthesis in various organs of dietary-restricted rats. Generation studies have shown that adaptation prevails in the first, second and third generation offspring of dietary-insulted rats, after which all parameters decline in fourth and fifth generation offspring. By reducing the litter size and exchanging the pups of control and dietary-restricted rats during the lactation period, partial restoration of the cellular growth of different organs is effected with the exception of the brain, in which damage is irreversible.     

甜瓜籽营养成分分析

为了进一步开发利用新疆的甜瓜籽资源,分析了新疆甜瓜籽的营养成分、氨基酸组成、微量元素的含量以及甜瓜籽油的理化指标和脂肪酸组成.结果表明,甜瓜籽含37.12%的油,24.08%的蛋白质,24.50%的糖类,0.010%的VE;甜瓜籽蛋白质中氨基酸种类齐全;甜瓜籽油富含不饱和脂肪酸,含量高达90%多;甜瓜籽油的理化指标为相对密度d20 4 0.9228,碘值174.56 gI/100g,折光指数n201.4589,皂化值195.32 mgKOH/g,不皂化物7.86%.