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81.
82.
A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways. 总被引:2,自引:1,他引:1
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J. A. Wilkins J. Cone Z. I. Randhawa D. Wood M. K. Warren H. E. Witkowska 《Protein science : a publication of the Protein Society》1993,2(2):244-254
The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates. 相似文献
83.
Stein and Moore Award address. The molten globule intermediate of apomyoglobin and the process of protein folding. 总被引:7,自引:6,他引:1
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The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of alpha-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. & Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N<-->I<-->U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. & Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., & Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagnesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., & Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
84.
85.
有机酸是含有一种或多种低分子量酸性基团(如羧基、磺酸基)的可生物合成的有机化合物,广泛应用于食品、农业、医药、生物基材料工业等领域。酵母菌具有生物安全、抗逆性强、底物谱广泛、方便遗传改造,以及大规模培养技术成熟等独特优点,因此利用酵母菌生产有机酸的研究日益受到国内外学者的关注。目前利用酵母生产有机酸还存在浓度低、副产物多,以及发酵效率低等缺陷。随着酵母菌代谢工程和合成生物学技术的发展,利用酵母菌生产有机酸取得了快速进展。本文总结了利用酵母合成11种有机酸的研究,包括内源和异源合成的大宗羧酸和高价值有机酸,并对该领域的未来研究方向进行了展望。 相似文献
86.
S-腺苷甲硫氨酸(S-adenosyl-l-methionine, SAM)广泛存在于生物体内,主要参与生物体内的转甲基过程、转硫过程及转氨丙基过程,具有重要的生理功能,其生产备受重视。目前SAM生产的研究主要集中于微生物发酵法,该方法与化学合成法和酶催化法相比,成本较低且更容易实现工业化生产。随着需求量的迅速增加,通过菌种改良提高SAM产量备受关注。当前SAM生产菌种改良的主要策略包括常规育种和代谢工程。本文综述了提高微生物生产SAM能力的近期研究进展并探讨了SAM生产中的瓶颈问题及解决方法,以期为进一步提高SAM产量提供思路。 相似文献
87.
L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。 相似文献
88.
Type 2 diabetes (T2D) and Alzheimer's disease (AD) are two global epidemics that share several metabolic defects, such as insulin resistance, impaired glucose metabolism, and mitochondrial defects. Importantly, strong evidence demonstrates that T2D significantly increases the risk of cognitive decline and dementia, particularly AD. Here, we provide an overview of the metabolic defects that characterize and link both pathologies putting the focus on mitochondria. The biomarker potential of mitochondrial components and the therapeutic potential of some drugs that target and modulate mitochondria are also briefly discussed. 相似文献
89.
Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri-, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus-1, and O2A from Operophtera brumata cypovirus-18. Applying the polycistronic-like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast. 相似文献
90.
William T. Scott David Henriques Eddy J. Smid Richard A. Notebaart Eva Balsa-Canto 《Biotechnology and bioengineering》2023,120(7):1998-2012
Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings. 相似文献