Systematic interpolation method predicts protein chromatographic elution with salt gradients,pH gradients and combined salt/pH gradients

A methodology is presented to predict protein elution behavior from an ion exchange column using both individual or combined pH and salt gradients based on high‐throughput batch isotherm data. The buffer compositions are first optimized to generate linear pH gradients from pH 5.5 to 7 with defined concentrations of sodium chloride. Next, high‐throughput batch isotherm data are collected for a monoclonal antibody on the cation exchange resin POROS XS over a range of protein concentrations, salt concentrations, and solution pH. Finally, a previously developed empirical interpolation (EI) method is extended to describe protein binding as a function of the protein and salt concentration and solution pH without using an explicit isotherm model. The interpolated isotherm data are then used with a lumped kinetic model to predict the protein elution behavior. Experimental results obtained for laboratory scale columns show excellent agreement with the predicted elution curves for both individual or combined pH and salt gradients at protein loads up to 45 mg/mL of column. Numerical studies show that the model predictions are robust as long as the isotherm data cover the range of mobile phase compositions where the protein actually elutes from the column.     

Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.

The technique developed by Casadaban (M. J. Casadaban, J. Mol. Biol. 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon. Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated. When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium. Repressing conditions prevent growth on lactose. This property of bio-lac fusion strains, as well as the ease of determining the level of operon expression by assaying beta-galactosidase, was used for the isolation and characterization of mutants defective in repression. Preliminary analyses of several newly isolated regulatory mutants are presented. For the several birA mutants examined, there appeared to be no direct correlation between effects on minimum biotin requirement and alterations in repressibility, suggesting a possible dual function for the gene. Parallel attempts to obtain fusions of lac to bioH were unsuccessful, indicating lack of direct biotin control at the bioH locus.     

Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.     

Multicopy plasmids affect replisome positioning in Bacillus subtilis

The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。     

RevSex duplication-induced and sex-related differences in the SOX9 regulatory region chromatin landscape in human fibroblasts

It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of “chromatin-CGH”) and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14–15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.     

Characterization and identification of the inhibitory domain of GDF-8 propeptide

GDF-8 is a negative regulator of skeletal muscle mass. The mechanisms which regulate the biological activity of GDF-8 have not yet been elucidated. Analogous to the TGF-beta system, GDF-8 propeptide binds to and inhibits the activity of GDF-8. In these studies, we define the critical domain of the GDF-8 propeptide necessary for inhibitory activity. Two molecules of GDF-8 propeptide monomer inhibit the biological activity of one molecule of GDF-8 homodimer. Although the propeptide contains N-linked glycosylation when synthesized in mammalian cells, this glycosylation is not necessary for the inhibition of GDF-8. Taking advantage of the bacterial expression system, we express and purify GDF-8 propeptide which retains full inhibitory activity. To define the functional regions of the propeptide, we express a series of truncated GST-propeptide fusion proteins and examined their inhibitory activity. We observe that fusion proteins containing the C-terminal region (amino acid residues 99-266) are very stable, but do not exhibit inhibitory activity; while fusion proteins containing the N-terminal region (amino acid residues 42-115) are labile but contain essential inhibitory activity. The data suggest that the C-terminal region may play a role in the stability of the GDF-8 propeptide and that the inhibitory domain is located in the region between amino acids 42 and 115.     

Differentially regulated expression of endogenous RGS4 and RGS7

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27-28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30-40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.