Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC₅₀ value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.     

Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae

A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis. A 5.3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.     

Construction of validated, non-redundant composite protein sequence databases

A strategy has been developed for the construction of a validated,comprehensive composite protein sequence database. Entries areamalgamated from primary source data bases by a largely automatedset of processes in which redundant and trivially differententries are eliminated. A modular approach has been adoptedto allow scientific judgement to be used at each stage of databaseprocessing and amalgamation. Source databases are assigned apriority depending on the quality of sequence validation andcommenting. Rejection of entries from the lower priority database,in each pairwise comparison of databases, is carried out accordingto optionally defined redundancy criteria based on sequencesegment mismatches. Efficient algorithms for this methodologyare embodied in the COMPO software system. COMPO has been appliedfor over 2 years in construction and regular updating of theOWL composite protein sequence database from the source databasesNBRF-PIR, SWISS-PROT, a GenBank translation retrieved from thefeature tables, NBRF-NEW, NEWAT86, PSD-KYOTO and the sequencescontained in the Brookhaven protein structure databank. OWLis part of the ISIS integrated data resource of protein sequenceand structure [Akrigg et al. (1988) Nature, 335, 745–746].The modular nature of the integration process greatly facilitatesthe frequent updating of OWL following releases of the sourcedatabases. The extent of redundancy in these sources is revealedby the comparison process. The advantages of a robust compositedatabase for sequence similarity searching and information retrievalare discussed.     

Effect of amidation on the foaming and physico-chemical properties of bovine serum albumin

Amidation of bovine serum albumin (BSA) was achieved by a water-soluble carbo-diimide, ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated reaction using ammonium chloride as the nucleophile. Partial and substantial amidation of 0.5% (w/v) BSA in 5.5 m ammonium chloride solution with 1 times 10-²mmol EDC over 120 min and 1 times 10^-1mmol EDC over 10 min respectively was achieved on a large scale using diafiltration for rapid termination of the reaction and purification. Residual ammonium chloride otherwise enhanced foaming properties. The amidated proteins were characterized by isoelectric focusing, electrophoresis and hydrophobicity and disulphide- and sulphydryl-group measurements. Compared with native BSA, partially amidated BSA (PA-BSA) produced enhanced foam expansion and foam stability values. This was attributed to minimal denaturation and to the presence of both acidic and basic components (pI range 5.25–7.50) within the single protein. In contrast, substantially amidated BSA (SA-BSA) (pI range 7–9.1) had similar foaming properties to those of the ultrafiltered BSA control which were slightly lower than those of native BSA. However SA-BSA interacted synergistically with native BSA producing enhanced foaming properties particularly at the 1:1 ratio through electrostatic interactions, conformational changes and increased hydrophobicity.     

Hydration of Whey Powders as Determined by Different Methods

Four methods for evaluating water hydration of 15 whey derivative powders were compared, and results are discussed with respect to the chemical composition of the powders. Hydration capacities between 0.21 and 4.64 mL water/g of powder were obtained, depending on the method used. The filtration/centrifugation method gave the highest hydration capacity, whereas the paste-water retention method gave the lowest. The Baumann test and the paste-water retention method were well correlated with protein and lactose content of the powders, enabling differentiation between hydration capacities of whey protein concentrates (35% proteins) and electrodialyzed whey powders (12% proteins). Reliable characterization of hydration required a combination of methods.     

马家沟芹菜加工下脚料蛋白营养价值的化学评价

在对马家沟芹菜加工下脚料的蛋白含量及其氨基酸组成进行分析的基础上,应用氨基酸评分法,对其营养价值进行全面评价和比较.结果表明:马家沟芹菜加工下脚料蛋白的必需氨基酸含量很丰富,第一限制性氨基酸为色氨酸;各种人体必需氨基酸与FAO/WHO提出的理想蛋白质模式进行比较,马家沟芹菜加工下脚料蛋白的IEAA为95.88,从氨基酸...     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Molecular recognition of DNA–protein complexes: A straightforward method combining scanning force and fluorescence microscopy

Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein–DNA complexes.     

Supramolecular Bioconjugates for Protein and Small Drug Delivery

Supramolecular conjugation techniques have been developed to produce novel nanosized systems by assembling materials with diverse physicochemical and biological features. These techniques have been adapted to obtain innovative bioconjugates to deliver drugs with poor biopharmaceutical properties and nano-devices with potential “theranostic” activity. Supramolecular drug delivery systems include polymer therapeutics such as drug–polymer bioconjugates, and colloidal carriers such as micelles, liposomes, polyplexes, and organic and inorganic nanoparticles. By virtue of their wide array of chemical composition and properties, polymers represent key elements for the construction of novel supramoelcular formulations. Polymer bioconjugation is a fledged technique for fabrication of protein–polymer conjugates. PEGylation, in particular, produces derivatives with enhanced pharmacokinetic, immunological, and stability properties as compared to the parent protein. Over the years, new methods have been set up to obtain site-directed polymer conjugation. In this review we report few grafting to and growing from PEGylation examples for the preparation of therapeutically effective protein bioconjugates. Supramolecular formulations with unique properties can be also obtained by assembling functional polymers, targeting agents, physicochemical modifiers, and biomodulators. These systems may be designed for disease tissue disposition and cell recognition/penetration. Cyclodextrins, for example, have been functionalized with polyethylene glycol and folic acid to produce tumor-targeted drug carriers. Interesting results have been obtained with this novel class of drug delivery systems. In addition, responsive polymers have been conjugated to gold nanoparticles to endow a new colloidal platform with triggerable cell disposition properties, which can be exploited either in biomedicine or diagnosis.