The crystal structure of human receptor protein tyrosine phosphatase kappa phosphatase domain 1

The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPkappa is a member of the R2A/IIb subfamily of RPTPs along with RPTPmu, RPTPrho, and RPTPlambda. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPkappa at 1.9 A. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed beta-sheets flanked by alpha-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPmu structure are all conserved, they are not involved in the protein-protein interaction in RPTPkappa. The N-terminal beta-strand, formed by betax association with betay, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPkappa structure forming a beta-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPkappa as a monomer. In this family the crystal structure of catalytically active RPTPmu D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPkappa is monomeric in solution and crystal structure.     

无细胞蛋白合成体系实现胰岛素原可溶性表达

胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。     

大肠杆菌细胞抽提物制备方案的简化与优化

无细胞蛋白表达系统是一种将目的蛋白在体外进行表达的新技术和新方法,已广泛应用到蛋白质组学、蛋白质结构和功能等领域的研究中。在无细胞蛋白表达系统中,细胞抽提物的制备是关键因素之一。通过对大肠杆菌细胞抽提物制备过程中离心速度、预孵化和透析等参数的考察,利用绿色荧光蛋白作为报告蛋白,可以得到一个细胞抽提物制备的简化方案。采用相对低的转速(12 000×g,10 min),简易空孵化即可制备出活性高的细胞抽提物,用于无细胞体系蛋白表达,其表达的绿色荧光蛋白产量为209μg/mL。与传统的大肠杆菌细胞抽提物S30相比较,新方案将使时间与成本节省62%,产量是传统方法的2.6倍,使无细胞蛋白表达技术的操作快速、高通量的优势更加明显。     

颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达

为高效表达颗粒裂解肽G13结构域并避免G13对宿主菌的毒性, 将人工合成的编码G13的基因片段, PCR扩增后克隆于原核表达载体pThioHisA中, 构建了重组表达载体pThioHisA-G13, 将其转化于大肠杆菌BL21(DE3)中, 经IPTG诱导表达融合蛋白Trx-G13, 表达产物以包涵体的形式存在, 其表达量约占细菌总蛋白的58%。包涵体蛋白经 8 mol/L尿素溶解后, 再经CNBr切割, 阳离子交换层析, 得到纯化的重组G13结构域。琼脂糖扩散法检测表明重组G13结构域多肽具有抗菌活性。     

Study of protein-protein interactions by fluorescence of tryptophan analogs: application to immunoglobulin G binding domain of streptococcal protein G

The protein-protein interaction system often contains many fluorophores that may significantly interfere with the quantitative determination of the binding abilities. To solve this perplexing problem, we biosynthetically incorporated the two tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into the immunoglobulin G (IgG) binding domain of streptococcal protein G (PGBD). The exclusive excitation and novel fluorescence changes in both the intensity and anisotropy are beneficial to reporting the details of the interactions between PGBD and the IgG fragments and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 microM for the binding of human Fc and 8.0 microM for mouse Fc. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the binding abilities and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex.     

Ribosome display: cell-free protein display technology.

Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. It uses the principle of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes), through the formation of stable protein-ribosome-mRNA (PRM) complexes. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA, which is then converted and amplified as DNA for further manipulation, including repeated cycles or protein expression. Ribosome display has a number of advantages over cell-based systems such as phage display; in particular, it can display very large libraries without the restriction of bacterial transformation. It is also suitable for generating toxic, proteolytically sensitive and unstable proteins, and allows the incorporation of modified amino acids at defined positions. In combination with polymerase chain reaction (PCR)-based methods, mutations can be introduced efficiently into the selected DNA pool in subsequent cycles, leading to continuous DNA diversification and protein selection (in vitro protein evolution). Both prokaryotic and eukaryotic ribosome display systems have been developed and each has its own distinctive features. In this paper, ribosome display systems and their application in selection and evolution of proteins are reviewed.     

Trinucleotide repeat expansions catalyzed by human cell-free extracts

Trinucleotide repeat expansions cause 17 heritable human neurological disorders. In some diseases, somatic expansions occur in non-proliferating tissues such as brain where DNA replication is limited. This finding stimulated significant interest in replication-independent expansion mechanisms. Aberrant DNA repair is a likely source, based in part on mouse studies showing that somatic expansions are provoked by the DNA repair protein MutSβ (Msh2-Msh3 complex). Biochemical studies to date used cell-free extracts or purified DNA repair proteins to yield partial reactions at triplet repeats. The findings included expansions on one strand but not the other, or processing of DNA hairpin structures thought to be important intermediates in the expansion process. However, it has been difficult to recapitulate complete expansions in vitro, and the biochemical role of MutSβ remains controversial. Here, we use a novel in vitro assay to show that human cell-free extracts catalyze expansions and contractions of trinucleotide repeats without the requirement for DNA replication. The extract promotes a size range of expansions that is similar to certain diseases, and triplet repeat length and sequence govern expansions in vitro as in vivo. MutSβ stimulates expansions in the extract, consistent with aberrant repair of endogenous DNA damage as a source of expansions. Overall, this biochemical system retains the key characteristics of somatic expansions in humans and mice, suggesting that this important mutagenic process can be restored in the test tube.     

Combinatory use of cell-free protein expression,limited proteolysis and mass spectrometry for the high-throughput protein domain identification

The structural domains of proteins have often been identified through the use of limited proteolysis. In structural genomics studies, it is necessary to carry this out in a high-throughput manner. Here, we constructed a novel high-throughput system, which consists of cell-free protein expression and one-step affinity purification, followed by limited proteolysis using a unique new method, referred to “on beads method”. All these steps were carried out on 96-well plate formats and completed in two days, even by manual handling. The merits of the new method versus the conventional one are as follows: (1) experimental times are reduced, (2) the sample preparation for limited proteolysis experiments is simplified, and (3) both protein purification and limited digestion can be performed “in situ” on the same sample plate. This preparation method is therefore suitable for highly automated, proteolytic analyses coupled to mass spectrometry techniques at a micro-scale protein expression level. The resulting protease-resistant fragments were analyzed by MALDI-TOF-MS and protein domains of 34 mouse cDNA products were identified with this system.     

颗粒裂解肽G13结构域的重组表达及蛋白质结构预测

基因工程构建表达是获得抗菌肽的一种成本较低的方法,本实验人工合成G13结构域编码DNA序列,PCR扩增后,用T-A克隆法与pBAD/TOPO ThioFusion表达载体连接,通过PCR鉴定筛选出正确重组质粒,在大肠杆菌Top10中对目的蛋白进行表达,大肠杆菌工程菌经阿拉伯糖诱导后取样,用SDS-PAGE检测表达情况,采用生物信息学方法对表达蛋白的结构特征进行模拟分析。结果显示：目的蛋白在原核系统中实现了高效表达,表达量高达67%以上,主要以包涵体形式表达。蛋白结构预测结果显示,目的蛋白原有的α螺旋活性结构无改变,从而为抗菌肽高效生产提供了有效可靠的研究途径。     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

The evolutionary analysis reveals domain fusion of proteins with Frizzled-like CRD domain

Frizzleds (FZDs) are transmembrane receptors in the Wnt signaling pathway and they play pivotal roles in developments. The Frizzled-like extracellular Cysteine-rich domain (Fz-CRD) has been identified in FZDs and other proteins. The origin and evolution of these proteins with Fz-CRD is the main interest of this study. We found that the Fz-CRD exists in FZD, SFRP, RTK, MFRP, CPZ, CORIN, COL18A1 and other proteins. Our systematic analysis revealed that the Fz-CRD domain might have originated in protists and then fused with the Frizzled-like seven-transmembrane domain (7TM) to form the FZD receptors, which duplicated and diversified into about 11 members in Vertebrates. The SFRPs and RTKs with the Fz-CRD were found in sponge and expanded in Vertebrates. Other proteins with Fz-CRD may have emerged during Vertebrate evolution through domain fusion. Moreover, we found a glycosylation site and several conserved motifs in FZDs, which may be related to Wnt interaction. Based on these results, we proposed a model showing that the domain fusion and expansion of Fz-CRD genes occurred in Metazoa and Vertebrates. Our study may help to pave the way for further research on the conservation and diversification of Wnt signaling functions during evolution.     

绿色荧光蛋白GFP基因与新生隐球菌荚膜基因的融合表达系统

目的构建新生隐球菌荚膜基因与绿色荧光蛋白的融合表达系统。方法PCR法扩增CAP60基因片段,测序验证其准确性。将其与多个必需基因共同连人穿梭质粒。结果获得6150bps大小的质粒,该质粒含有荚膜基因启动子、终止子及荧光蛋白的基因。结论将新生隐球菌荚膜基因与荧光蛋白基因融合表达,将会有利于对荚膜的生化合成途径作进一步研究。     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Ugo1p is a multipass transmembrane protein with a single carrier domain required for mitochondrial fusion

The outer mitochondrial membrane protein Ugo1 forms a complex with the Fzo1p and Mgm1p GTPases that regulates mitochondrial fusion in yeast. Ugo1p contains two putative carrier domains (PCDs) found in mitochondrial carrier proteins (MCPs). Mitochondrial carrier proteins are multipass transmembrane proteins that actively transport molecules across the inner mitochondrial membrane. Mitochondrial carrier protein transport requires functional carrier domains with the consensus sequence PX(D/E)XX(K/R). Mutation of charged residues in this consensus sequence disrupts transport function. In this study, we used targeted mutagenesis to show that charge reversal mutations in Ugo1p PCD2, but not PCD1, disrupt mitochondrial fusion. Ugo1p is reported to be a single-pass transmembrane protein despite the fact that it contains several additional predicted transmembrane segments. Using a combination of protein targeting and membrane extraction experiments, we provide evidence that Ugo1p contains additional transmembrane domains and is likely a multipass transmembrane protein. These studies identify PCD2 as a functional domain of Ugo1p and provide the first experimental evidence for a multipass topology of this essential fusion component.     

Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities

"Host-guest" studies of the B1 domain from Streptococcal protein G have been used previously to establish a thermodynamic scale for the beta-sheet-forming propensities of the 20 common amino acids. To investigate the contribution of side chain conformational entropy to the relative stabilities of B1 domain mutants, we have determined the dynamics of side chain methyl groups in 10 of the 20 mutants used in a previous study. Deuterium relaxation rates were measured using two-dimensional NMR techniques for 13CH2D groups. Analysis of the relaxation data using the Lipari-Szabo model-free formalism showed that mutations introduced at the guest position caused small but statistically significant changes in the methyl group dynamics. In addition, there was a low level of covariation of the Lipari-Szabo order parameters among the 10 mutants. The variations in conformational free energy estimated from the order parameters were comparable in magnitude to the variations in global stability of the 10 mutants but did not correlate with the global stability of the domain or with the structural properties of the guest amino acids. The data support the view that conformational entropy in the folded state is one of many factors that can influence the folding thermodynamics of proteins.     

HSP70 kinetics study by continuous observation of HSP-GFP fusion protein expression on a perfusion heating stage

The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.     

A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell‐free protein synthesis systems

Cell‐free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell‐free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re‐examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell‐free system with a 95% reduction in reagent costs (excluding cell‐extract, plasmid, and T7 RNA polymerase made in‐house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze‐thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high‐throughput screening, and biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:823–831, 2015     

Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans

Clostridium cellulovorans produces a cellulase complex (cellulosome) as well as noncellulosomal cellulases. In this study, we determined a factor that affected the solubility of the cellulosomal cellulase EngB and the noncellulosomal EngD when they were expressed in Escherichia coli. The catalytic domains of EngB and EngD formed inclusion bodies when expressed in E. coli. On the other hand, both catalytic domains containing the C-terminal cellulose-binding domain (CBD) of EngD were expressed in soluble form. Fusion with the CBD of EngD also helped increased the solubility of cellulosomal cellulase EngL upon expression in E. coli. These results indicate that the CBD of EngD plays an important role in the soluble expression of the catalytic domains of EngB, EngL, and EngD. The possible mechanisms of solubilization by fusion of the catalytic domain with the CBD from EngD are discussed.     

Influence of hPin1 WW N-terminal domain boundaries on function, protein stability, and folding

An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.