Engineering of stable and fast-folding sequences of model proteins.

The statistical mechanics of protein folding implies that the best-folding proteins are those that have the native conformation as a pronounced energy minimum. We show that this can be obtained by proper selection of protein sequences and suggest a simple practical way to find these sequences. The statistical mechanics of these proteins with optimized native structure is discussed. These concepts are tested with a simple lattice model of a protein with full enumeration of compact conformations. Selected sequences are shown to have a native state that is very stable and kinetically accessible.     

日本血吸虫未知基因的原核表达载体的构建和表达分析

目的构建日本血吸虫未知基因的原核表达载体,研究基因性质. 方法首先将从日本血吸虫成虫cDNA文库中筛选得到的未知基因cDNA JAYL0230亚克隆入原核表达载体pET28a(+)中,IPTG诱导重组蛋白的表达,免疫印记实验检测其免疫性质. 结果成功构建重组原核表达载体pET28a(+)-JAYL0230,并获得稳定表达的融合蛋白,免疫印记实验证明该融合蛋白具有免疫反应性. 结论本实验为进一步深入研究该基因的性质及功能提供了基础.     

巴豆提取物诱导小鼠小肠组织中蛋白质差异表达的初步研究

目的 建立和优化蛋白质分子差异展示的双向聚丙烯酰胺凝胶电技术,初步观察巴豆提取物诱导小鼠小肠组织中蛋白质的差异表达,为进一步筛选其中介导巴豆生物作用的蛋白质分子奠定基础。方法 应用巴豆提取物灌胃制备小刀慢有肠运动增强模型,提取其小肠组织中总蛋白质,优化蛋白质分子差异展示的双向聚丙安凝胶电泳技术,并用其分析模型动物小肠组织中蛋白质,双向电泳凝胸银染法显示其蛋白质的差异表达。结果 建立了小鼠慢性胃肠运     

Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20–30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE.Laemmli’s system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences (1). The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in “overnight” adoption of the technique in biochemistry, cell biology, immunology, and virology (2). Considered “the single most useful analytical tool to study protein molecules” (3), SDS/PAGE is routinely used for simultaneous determination of protein heterogeneity and molecular weight in applications ranging from diagnosis of hereditary red cell membrane disorders to evaluation of recombinant protein expression and purification procedures. Protein analysis by SDS/PAGE is relatively simple, affordable, and rapid (4): A buffer containing a tracking dye and SDS is added to the sample of interest, the mixture is applied to a polyacrylamide gel, and a potential difference is used to drive the dye and the resulting anionic particle composed of protein and dodecyl sulfate (DS) through the gel. The distance traveled by the protein/DS particle from the top of the gel is then divided by that of the dye to obtain relative migration (R_f), and molecular weight [as relative molecular mass (M_r)] determined by comparison of this value with a logarithmic plot derived from the R_fs and M_rs of reference proteins.Fractionation on SDS/PAGE is controlled by the molecular size and shape of the protein/DS particle, its net charge, and the accessible spaces among the acrylamide fibers that comprise the gel matrix as determined by the total concentration of acrylamide and bis-acrylamide cross-linker [T; Materials and Methods (5)]. Larger particles become trapped within the gel meshwork and migrate slower than smaller species. Low-percentage gels are therefore typically used to resolve larger proteins, and vice versa. Acrylamide concentrations compatible with routine use are usually from 4–20% T due to practical considerations, because gels outside of this range are too fragile or too brittle, respectively, to withstand the physical manipulation(s) required for protein visualization and/or immunoblotting.Most globular, water-soluble proteins are reliably identified by their SDS/PAGE mobility relative to corresponding reference proteins typically used to estimate molecular weight. However, this group of well-behaved polypeptides does not include helical transmembrane (TM) proteins, macromolecules that comprise 20–30% of the human genome (6), comprise the majority of drug targets (7), and are the focus of major pharmaceutical discovery efforts (8). For example, the first true G protein-coupled receptor to be determined to high resolution, 39-kDa bovine rhodopsin (9), migrates on SDS/PAGE to positions consistent with sizes as low as 30 kDa (10). In fact, we have previously shown that the gel mobility of helical TM proteins seldom corresponds to formula molecular weight (11). This phenomenon of “anomalous migration” can arise as a consequence of the high hydrophobicity and concomitant binding of DS by TM proteins at levels that exceed those of water-soluble polypeptides (12). However, quantitation of DS binding stoichiometry is not routine and consumes milligram amounts of purified samples. Thus, the impact of enhanced DS binding on the direction and magnitude of anomalous migration has remained unpredictable for decades, with helical TM proteins variously exhibiting gel mobility reduced, equivalent, or increased relative to reference proteins (11–13). Such differences are generally disregarded when protein identity is known or can be confirmed in orthogonal molecular weight determination procedures but, in many instances, raise questions of protein folding, oligomeric organization, proteolytic processing, posttranslational modification(s), alternative splicing, antibody cross-reaction, and/or degradation. These issues become acute in SDS/PAGE analyses of tissue or cell extracts, where reasonable molecular weight estimates remain crucial for protein identification.Here, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of de novo designed TM-mimetic peptide polymers with typical water-soluble reference proteins on Laemmli gels ranging from 11–18% T. We find that net charge and effective molecular size among the migrating TM-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms derived from these data compensate for the differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a straightforward means to predict anomalous migration of membrane proteins relative to reference polypeptides, facilitating their identification by molecular weight in SDS/PAGE applications.     

促肝细胞生长素及其类似物的生物活性研究

目的 研究促肝细胞生长素(pHGF)对肝细胞及肝星状细胞生物活性的影响。方法 以人肝细胞株及大鼠肝星状细胞株为研究靶细胞,采用MTT法研究pHGF对细胞增殖活性的影响,放免法检测透明质酸(HA),细胞基因转染结合报告基因测活检测Ⅰ型胶原基因转录活性的变化,SDS-PAGE分析pHGF主要成分。结果 pHGF具有明确促进肝细胞增生及抑制肝纤维化主要形成细胞肝星状细胞系增殖的作用,能抑制细胞外间质成分HA的产生,抑制Ⅰ型胶原基因启动子样活性。研究的12种pHGF中仅1种在15％SDS-PAGE中具有明确的蛋白条带。结论 pHGF具有促进肝细胞再生及抗纤维化作用,结构-功能关系需进一步研究。     

Human growth hormone (GH)-releasing factor stimulates and somatostatin inhibits the release of rat GH variants

Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was used for the analysis of proteins secreted by male rat pituitary cells in monolayer culture in the presence of 10 nM human GH-releasing factor (hGRF) or 30 nM somatostatin (SRIF) or in the absence of these factors. More than 300 medium proteins were reproducibly detected either by fluorographic autoradiography or by silver staining. Immunoreactivity of each protein was examined after 2D PAGE followed by Western blotting and immunostaining with affinity-purified antirat GH (rGH) antibody. While there was a cluster of immunoreactive spots in the GH dimer range (40,000-50,000 mol wt), at least 16 medium proteins of mol wt 22,000 or less were also stained. Among these 16 proteins the release of 15 was stimulated and the release of 14 was inhibited by hGRF and SRIF, respectively. On the other hand, there were 3 proteins of approximate mol wt 16,000 whose secretion was regulated in a coordinate manner as rGH by the hypothalamic factors but which did not cross-react with anti-rGH antibodies. The increase or decrease in the radioactivity of each protein spot obtained from media after pituitary cells were incubated with [35S]methionine and hypothalamic factors was analyzed statistically. A pulse-chase study suggested that at least 7 of the hormonally regulated proteins, including rGH, were synthesized very rapidly. Finally, the 2D PAGE analysis of cell-free translation products of messenger RNA derived from male rat anterior pituitaries revealed the presence of about 40 rGH-immunoreactive proteins which included pre-GH. These data suggest that there are multiple forms of rGH-variants or rGH-related proteins. The biological significance(s) of all the rGH immunoreactive proteins and of the GRF- and SRIF-regulated pituitary proteins remains unclear. It is evident that a number of these proteins are synthesized and released rapidly by pituitary cells in culture. Furthermore, the presence of multiple genes for these rGH-related proteins is suggested by the large family of immunoreactive gene products identified after cell-free translation of messenger RNA derived from the pituitary.     

Palmitoylation of platelet proteins

Palmitoylation as incorporation of [(3)H] palmitic acid into proteins occurred in platelets as in other cell systems. The linkage of palmitic acid to platelet proteins was stable to SDS and organic solvents but was sensitive to hydroxylamine, consistent with oxyester or thioester bond(s). Non-reduced SDS PAGE analysis revealed that the most prominantly labelled proteins were a quadruplet of proteins of 30-38 kDa. Under reducing conditions, these proteins appeared as a doublet of 38 kDa proteins. Thrombin or PMA enhanced the incorporation of label into these proteins, suggesting the involvement of palmitoylation in platelet activation. In prelabelled platelets, A23187 led to a E-64d-sensitive decrease of label associated with a 35 kDa protein while PMA caused a stuarosporine-sensitive shift of the same protein in a non-reduced gel. Some of the palmitoylated proteins were associated with the cytoskeleton. Thrombin appeared to have different effects on these labelled cytoskeletal proteins. While the identity of most of the palmitoylated platelet proteins remains unknown, a few palmitoylated proteins have been identified to be CD9, glycoproteins IIIa, Ib and IX.     

生长抑素受体2转染胰腺癌细胞生物学行为的研究

目的探讨生长抑素受体2(SSTR2)对胰腺癌恶性行为的影响。方法利用原先已构建的腺病毒载体Adv-GFP-SST2R将人SSTR2全长cDNA转染导入胰腺癌细胞株PC3中,用RT-PCR检测SSTR2mRNA表达。通过肿瘤细胞与基膜成分黏附能力测定、运动能力测定、明胶酶谱法观察转染前后细胞的生物学特性的改变。结果转染Adv-GFP-SST2R的PC3细胞表达SST2RmRNA。SSTR2转染胰腺癌PC3细胞后,胰腺癌细胞与基质的黏附性、运动性下降,明胶酶的分泌能力明显减弱。结论SSTR2转染可明显减轻胰腺癌的恶性行为。     

细粒棘球蚴囊液蛋白组分分析

目的　利用鸟枪法分析细粒棘球蚴（Echinococcus granulosus）囊液蛋白（hydatid cyst fluid protein,HCFP）组分,寻找其中具有潜在调控免疫失调性疾病功能的活性组分。方法　无菌收集细粒棘球蚴病患者肝囊肿中的细粒棘球蚴囊液,采用鸟枪法进行液相色谱⁃串联质谱鉴定,采用MaxQuant 1.6.1.0软件进行数据库检索。利用基因本体论（Gene Ontology,GO）对已鉴定的蛋白质从细胞组分、分子功能和生物学功能三个方面进行分类。结果　经十二烷基磺酸钠⁃聚丙烯酰胺凝胶电泳（SDS⁃PAGE）分离的细粒棘球蚴囊液蛋白条带相对分子量为25～70 kDa,质谱分析共获得37个蛋白,其中已鉴定蛋白32种、未知蛋白5种。已初步发现至少有4种蛋白具有潜在调节免疫失调性疾病作用,即抗原B、谷胱甘肽S⁃转移酶、硫氧还蛋白过氧化物酶和苹果酸脱氢酶。GO富集分析显示,已鉴定蛋白具有149种分子功能,参与341种生物学过程。结论　细粒棘球蚴囊液蛋白成分复杂多样,从已知蛋白中初步筛选出4种与调节免疫失调性疾病潜在相关的蛋白。     

Amino acid residue at position 13 in HLA-DR beta chain plays a critical role in the development of Kaposi's sarcoma in AIDS patients

BACKGROUND: In 1994 human herpesvirus 8 (HHV-8) was identified as the causative agent of Kaposi's sarcoma (KS). Moreover, the crucial role of HLA molecules in determining susceptibility to several infections was recognized. OBJECTIVES: To evaluate the influence of HLA-DRB1 polymorphism in KS susceptibility among HHV-8 infected AIDS patients. DESIGN: A matched case-control study was designed to identify possible biological and environmental risk factors for HIV associated KS. Cases were defined as any AIDS patient with a clinical diagnosis of KS and controls as any AIDS patient with an indicative disease other than KS or with CD4 cells counts < 200 x 10 cells/l, diagnosed at +/- 4 months after case diagnosis. Each case was matched with two controls by sex, age and transmission category. METHODS: HHV-8 serostatus was determined by immunofluorescence assay for the latency associated antigen encoded by Orf73, ELISA for Orf73 and ELISA for the lytic antigen Orf65. DRB1 typing was carried out with a commercially available PCR-sequence specific primer assay. RESULTS: Comparison of marker frequencies in HHV-8 infected AIDS patients with or without KS showed a positive association between KS and HLA-DRB1 alleles containing phenylalanine at position 13 [odds ratio (OR), 2.24; P = 0.016]. A negative association was observed when the residue at the same position was glycine (OR, 0.16; P = 0.009). CONCLUSION: These observations suggest a possible role for HLA-DRB1 in the development of KS in HHV-8 infected individuals with HIV co-infection. Progression to KS in HHV-8 infected AIDS patients may also depend on host factors controlling the immune response.     

Purification and characterization of the estrogen-induced protein (IP) of the rat uterus

One of the earliest responses of the rat uterus to estrogen is increased synthesis of a specific cytosol protein (IP). This synthesis is detectable within 40 min and is dependent on an even earlier actinomycin D-sensitive function. IP has now been purified to homogeneity as determined by SDS polyacrylamide gel electrophoresis (SDS PAGE). The procedure was complicated by a tendency of the more homogeneous and concentrated material to aggregate. Purification consisted of sequential chromatography, in the presence of 0.1% triton X-100, via DEAE-cellulose (2X), hydroxylapatite and agarose-acrylamide. These were followed by preparative PAGE and finally SDS PAGE. Molecular weight determination by Ferguson plot analysis yielded an apparent molecular weight of 45 000. On final SDS PAGE, the material consisted of two major bands: the 45 000 molecular weight IP band and a band with an estimated molecular weight of 80 000. This second band displayed an elevated synthesis in E₂-stimulated uteri similar to IP and appeared to consist of some form of aggregated IP. Carbohydrate determination on SDS gels using periodic acid-Schiff (PAS) stain was negative.Co-purification of labeled cytosol proteins from uteri of control and E₂-stimulated rats revealed that IP is synthesized to some extent in the unstimulated animal as well as the stimulated.     

Binding of synovial fluid proteins to monosodium urate crystals in vitro

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blot techniques were used to analyze proteins adsorbed in vitro to synthetic monosodium urate crystals (MSUC) from a variety of gouty biologic fluids. Distinct differences in adsorbed proteins were found in comparing synovial fluid (SF) with serum or plasma, particularly at 220 kD and below 19 kD. Further studies should consider the importance of the synovial milieu. Patterns of proteins adsorbed to MSUC from the SF of all 12 patients with gout were similar. A considerable number of polypeptides appeared to be selectively adsorbed. Of these, polypeptides associated with fibronectin, C1q and IgM were identified by immunotransblotting. The experiments also demonstrated that the overall polypeptide patterns obtained by SDS PAGE of eluates from MSUC exposed to SF were unaffected by heating MSUC to 180 degrees C, as well as by the volume of SF in the incubation mixture.     

日本血吸虫成虫肠道内容物中金属蛋白酶的鉴定

目的　鉴定日本血吸虫成虫肠道内容物中的金属蛋白酶。　方法　以明胶作底物 ,利用明胶 SDS PAGE分离日本血吸虫成虫肠道内容物 ,将电泳后的凝胶于不同 pH缓冲液和酶抑制剂中进行孵育 ,对其中的金属蛋白酶进行分析和鉴定。　结果　日本血吸虫成虫肠道内容物中存在降解明胶的金属蛋白酶 ,其最适 pH值为 7～ 9。金属蛋白酶抑制剂EDTA抑制其活性。电泳分离获得有活性的酶蛋白 ,该酶是血吸虫感染血清识别的弱抗原。　结论　日本血吸虫成虫肠道内容物存在金属蛋白酶     

Attempts to restore scrapie prion infectivity after exposure to protein denaturants.

A wealth of experimental evidence argues that infectious prions are composed largely, if not entirely, of the scrapie isoform of the prion protein. We attempted to restore scrapie infectivity after exposure to protein denaturants including urea, chaotropic salts, and SDS. None of the procedures restored infectivity. Dialysis to remove slowly chaotropic ions and urea failed to restore scrapie infectivity. Attempts to create monomers of the scrapie isoform of the prion protein under nondenaturing conditions using a wide variety of detergents have been unsuccessful, to date, except for one report claiming that scrapie infectivity could be recovered from 12% polyacrylamide gels after SDS/PAGE [Brown, P., Liberski, P. P., Wolff, A. & Gajdusek, D. C. (1990) Proc. Natl. Acad. Sci. USA 87, 7240-7244]. We found that < 0.001% of the infectious prion titer could be recovered from the region of a polyacrylamide gel where the denatured proteinase K-resistant core of the scrapie isoform of the prion protein and other 30-kDa proteins migrate. We conclude that under the denaturing conditions used for SDS/PAGE, the scrapie isoform of the prion protein is denatured and little or no renaturation occurs upon injection of fractions eluted from gels into animals for bioassays.     

A chromatographically purified human TGF-β1 fraction from virally inactivated platelet lysates

Background and Objectives TGF‐β1 exerts important physiological functions in osteogenesis and chondrogenesis and may be of therapeutic interest. The aim of this work was to develop a scalable purification process of TGF‐β1 from virally inactivated human platelets. Study Design and Methods Apheresis platelet concentrates (N = 12) were solvent/detergent (S/D) treated (1% TnBP/1% Triton X‐45; 31 °C) and the resulting platelet lysates were clarified by oil extraction and centrifugation, then chromatographed on an anion‐exchange DEAE‐Sepharose Fast‐Flow column equilibrated in a PBS buffer, pH 7·5. The column was washed to eliminate unbound proteins and the S/D agents. Bound proteins were eluted using a 1 M NaCl–PBS buffer pH 7·5 (DEAE‐eluate). The content in TGF‐β1, PDGF‐AB, VEGF, IGF‐1, EGF, and b‐FGF was measured by ELISA. Proteins, lipids, and S/D agents were assessed. Protein profile was determined by SDS‐PAGE under reduced or non‐reduced conditions. Results Most proteins, including albumin and immunoglobulins G, A, and M did not bind to the DEAE column as evidenced also by SDS‐PAGE. Essentially all PDGF, VEGF, and IGF were in the breakthrough. The DEAE‐eluate contained close to 60% of the TGF‐β1 at a mean concentration of about 102 ng/ml, whereas EGF, b‐FGF were at about 0·72 and 0·18 ng/ml, respectively. The content in TnBP and Triton X‐45 was <2 ppm. Conclusion A fraction enriched in TGF‐β1 can be prepared from virally inactivated human platelet lysates using an easily scale process. Its interest in regenerative medicine and cell therapy will be evaluated in further studies.     

Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for 125I-labeled human choriogonadotropin, with an equilibrium dissociation constant (Kd) of 2.5 x 10(-9) M, which is comparable to the Kd values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. These two bands were also detected in subsequent direct ligand blotting analysis when the purified receptor was electrophoretically transferred to a nitrocellulose membrane after SDS/PAGE under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the Mr 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified Mr 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the Mr 135,000 species was almost completely converted to a Mr 92,000 band, but the Mr 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked Mr 92,000 subunit, which comprises the hormone-binding domain.     

Levels of keratan sulfate in the serum and synovial fluid of patients with osteoarthritis of the knee

We examined the relationship between serum and synovial fluid (SF) levels of antigenic keratan sulfate (KS) and the clinical, laboratory, and radiologic features of disease in 125 well-characterized patients with knee osteoarthritis (OA). KS was quantified by enzyme-linked immunosorbent assay, using an antibody specific for a highly sulfated epitope on KS chains; the results were calculated as equivalents of an international standard of KS from human costal cartilage. The mean level of serum KS (393 ng/ml) was significantly higher than those previously reported for populations of adults without OA. There was a wide scatter of serum KS values (range 156-912 ng/ml), with little correlation with clinical or radiologic features. Men had significantly higher levels than women (456 +/- 135 ng/ml versus 368 +/- 110 ng/ml, mean +/- SD), and there was a statistically significant but weak association with indicators of polyarticular involvement (number of symptomatic joints, Heberden's nodes, hip symptoms) in women. Despite the wide scatter of results in the population as a whole, individual levels of KS were stable for up to 4 consecutive years in the 9 patients studied. Levels of KS were much higher in SF (n = 25) than in serum, but the two were not correlated. There was an inverse correlation between radiographic evidence of cartilage loss and the level of KS in SF. The large variations in serum KS values suggest that this measure may not be of diagnostic significance among populations of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA energy landscapes via calorimetric detection of microstate ensembles of metastable macrostates and triplet repeat diseases

Biopolymers exhibit rough energy landscapes, thereby allowing biological processes to access a broad range of kinetic and thermodynamic states. In contrast to proteins, the energy landscapes of nucleic acids have been the subject of relatively few experimental investigations. In this study, we use calorimetric and spectroscopic observables to detect, resolve, and selectively enrich energetically discrete ensembles of microstates within metastable DNA structures. Our results are consistent with metastable, “native” DNA states being composed of an ensemble of discrete and kinetically stable microstates of differential stabilities, rather than exclusively being a single, discrete thermodynamic species. This conceptual construct is important for understanding the linkage between biopolymer conformational/configurational space and biological function, such as in protein folding, allosteric control of enzyme activity, RNA and DNA folding and function, DNA structure and biological regulation, etc. For the specific DNA sequences and structures studied here, the demonstration of discrete, kinetically stable microstates potentially has biological consequences for understanding the development and onset of DNA expansion and triplet repeat diseases.     

Polymorphism in the Mr 32,000 Rh protein purified from Rh(D)-positive and -negative erythrocytes.

A Mr 32,000 integral membrane protein has previously been identified on erythrocytes bearing the Rh(D) antigen and is thought to contain the antigenic variations responsible for the different Rh phenotypes. To study it on a biochemical level, a simple large-scale method was developed to purify the Mr 32,000 Rh protein from multiple units of Rh(D)-positive and -negative blood. Erythrocyte membrane vesicles were solubilized in NaDodSO4, and a tracer of immunoprecipitated 125I surface-labeled Rh protein was added. The Rh protein was purified to homogeneity by hydroxylapatite chromatography followed by preparative NaDodSO4/PAGE. Approximately 25 nmol of pure Rh protein was recovered from each unit of Rh(D)-positive and -negative blood. Rh protein purified from both Rh phenotypes appeared similar by one-dimensional NaDodSO4/PAGE, and the N-terminal amino acid sequences for the first 20 residues were identical. Rh proteins purified from Rh(D)-positive and -negative blood were compared by two-dimensional iodopeptide mapping after 125I-labeling and alpha-chymotrypsin digestion. The peptide maps were very similar; however, at least two additional iodopeptides were consistently noted in the Rh proteins purified from Rh(D)-positive erythrocytes. These data indicate that a similar core Rh protein (or group of related proteins) exists in both Rh(D)-positive and -negative erythrocytes, and the Rh proteins from erythrocytes with different Rh phenotypes contain distinct structural polymorphisms.     

Design and construction of diverse mammalian prion strains

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.