Plasma desorption mass spectrometry, an analytical tool in protein engineering: characterization of modified insulins

Californium-252 plasma desorption mass spectrometry (PDMS) hasbeen employed for the characterization of a series of humaninsulin derivatives in order to evaluate the performance ofthis technique as an analytical tool in protein engineering.Several of the characterized modifications result in a 1 a.m.u.mass change. The precision in mass determination obtainableby PDMS analysis is not sufficient for unambiguous verificationof such modifications based on the molecular weight alone. Itis, however, possible to carry out in situ enzymatic digestionof the sample. Subsequent PDMS analysis will in most cases revealif the modification has been introduced as intended.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminalextracellular domains of human CD4 antigen, a cell surface glycoprotein,is reported. This region of CD4, particularly the first domain,has been identified as containing the binding region for theenvelope gp120 protein of the human immuno-deficiency virus.The model was predicted based on the sequence homology of eachdomain with the variable light chain of immunoglobulins. Theframework ß-sheet regions were taken from the crystalcoordinates of REI. For one region in the first domain of CD4there was an ambiguity in the alignment with REI and two alternatemodels are presented. Loops connecting the framework were modeledfrom fragments selected from a database of main chain coordinatesfrom all known protein structures. Residues identified as involvedin binding gp120 have been located in several other studieswithin the first domain of CD4. Epitopes from eight monoclonalantibodies have been mapped onto residues in both domains. Competitionof these antibodies with each other and with gp120 can be interpretedfrom the structural model.     

The elusive role of the N-terminal extension of {beta}A3- and {beta}Al-crystallin

ß-Crystallins are structural lens proteins with aconserved two-domain structure and variable N- and C-terminalextensions. These extensions are assumed to be involved in quaternaryinteractions within the ß-crystallin oligomers orwith other lens proteins. Therefore, the production of ßA3-and ßAl-crystallin from the single ßA3/A1mRNA by dual translation initiation is of interest. These crystallinsare identical, except that ßAl has a much shorterN-terminal extension than ßA3. This rare mechanismhas been conserved for over 250 million years during the evolutionof the ßA3/A1 gene, suggesting that the generationof different N-terminal extensions confers a selective advantage.We therefore compared the stability and association behaviourof recombinant ßA3- and ßAl-crystallin.Both proteins are equally stable in urea- and pH-induced denaturationexperiments. Gel filtration and analytical ultracentrifugationestablished that ßA3 and ßA1 both form homodimers.In the water-soluble proteins of bovine lens, ßA3and ßA1 are present in the same molecular weight fractions,indicating that they oligomerize equally with other ß-crystallins.¹H-NMR spectroscopy showed that residues Met1 to Asn22 of theN-terminal extension of ßA3 have great flexibilityand are solvent exposed, excluding them from protein interactionsin the homodimer. These results indicate that the differentN-terminal extensions of ßA3 and ßA1 donot affect their homo- or heteromeric interactions.     

Destabilizing interactions between the partners of a bifunctional fusion protein

Hybrid MalE–GVP is a bifunctional protein in vitro sinceit binds maltose as protein MalE of Escherichia coli and sinceit is dimeric and specifically binds single-stranded DNA asprotein GVP of phage M13. The oxidation rate of a unique cysteineresidue was used to compare the stabilities of GVP in its freeand hybrid forms, under conditions where MalE was either foldedor unfolded by a denaturing agent. The results showed that boththe covalent link and tertiary non-covalent interactions betweenMalE and GVP destabilized GVP in MalE–GVP. To test whetherGVP had identical structures in its free and hybrid forms, mutationswere used as local conformational probes. The effects of thesemutations on the capabilities of MalE–GVP to dimerizeand to bind single-stranded DNA were assayed in vitro. Theywere compatible with the effects of the same mutations on theglobal activity of free GVP in vivo and with the effects thatcould be predicted from the known data on free GVP, in particularits crystal structure. Thus, one partner of a hybrid proteincan be destabilized by the other partner while maintaining itsstructural and functional characteristics.     

Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans

Flaking and extruding dehulled soybeans were evaluated as a means of enhancing oil extraction efficiency during enzyme-assisted aqueous processing of soybeans. Cellulase, protease, and their combination were evaluated for effectiveness in achieving high oil extraction recovery from extruded flakes. Aqueous extraction of extruded full-fat soy flakes gave 68% recovery of the total available oil without using enzymes. A 0.5% wt/wt protease treatment after flaking and extruding dehulled soybeans increased oil extraction recovery to 88% of the total available oil. Flaking and extruding enhanced protease hydrolysis of proteins freeing more oil. Treating extruded flakes with cellulase, however, did not enhance oil extraction either alone or in combination with protease. Discrepancies in oil extraction recoveries were encountered when merely considering crude free fat because some oil became bound to denatured protein during extrusion and/or sample drying. Bound fat was unavailable for determination by using the hexane extraction method, but was accounted for by using the acid hydrolysis method for total oil determination. Oil extraction recovery from extruded soybean flakes was affected by oil determination methods, which was not the case for unextruded full-fat soy flour.     

绿脓杆菌外膜蛋白对感染的保护作用

目的 寻找一种防治绿脓杆菌感染的有效方法。方法 用绿脓杆菌外膜蛋白免疫的兔抗血清对4种不同血清型的菌株和1株临床绿脓杆菌进行体内外交叉保护性试验。结果 家兔免疫1周后即产生抗体,并逐渐升高,至第6周抗体滴度达到1:163 840,并维持高水平;抗血清能与4种不同血清型菌株和1株临床菌株产生直接凝集反应。第8周的血清(1:8以上)与其外胰蛋白的ELISA抗体效价相近,小鼠的半保护剂量均在0.05～0.11ml之间。结论 外膜蛋白疫苗具有较强的抗原性,而且具有良好的交叉保护作用。     

Increasing productivity and protein content using early-maturing rices and efficient nitrogen management

Germplasm with shorter duration than that of the currently grown varieties is being generated to maximize productivity of irrigated rice. However, short-duration varieties often produce yields lower than the medium- and long-duration varieties. Experiments were conducted during the 1980–82 dry and wet seasons to increase productivity through the use of very early-maturing rices and the improved management of nitrogen (N) fertilizers.Results over three years showed that IR58 and IR9729-67-3 (growth duration 100 ± 5 days) yield as well as or higher than IR36 although earlier maturing. They generally had a higher productivity (kg ha^–1 day^–1) than IR36 (110 ± 5 days).Three years' data suggest that the improved timing of broadcast applications of urea in split doses increased grain yield comparable with the basal incorporation of slow-release sulfur-coated urea (SCU) or deep point-placement of urea supergranules (USG).Results on elite breeding lines showed that the early-maturing IR9729-67-3 produced higher protein yield ha^–1 than longer duration varieties such as IR8 and IR42 in the dry season. Furthermore, contrary to earlier results, single basal incorporation of slow-release SCU increased the protein yield of rice by 53 kg ha^–1 and deep point-placement of USG by 43 kg ha^–1 over split application of prilled urea.     

低蛋白天然胶乳的研究现状

简述了低蛋白天然胶乳的发展、制备方法、热降解性能和应用，并对低蛋白胶乳中蛋白质含量的检测方法和低蛋白硫化胶乳胶膜的力学性能作了简要的阐述。     

Preparation and protein adsorption of hydrogel polysucrose microspheres

A series of novel hydrogel polysucrose microspheres with the mean size ranging from 200 to 500 μm were prepared via two‐step method. First, soluble polysucrose was synthesized by solution polymerization between sucrose and epichlorohydrin; second, a reversed phase suspension crosslinking reaction was performed to prepare polysucrose microspheres. The SEM images indicated that these spherical beads had smooth surface and hydrogel interior structure. FTIR was used to characterize the chemical structure of the beads. The hydrated and dry densities, equilibrium water content, and hydroxyl content of polysucrose microspheres were also investigated. The characteristic of high hydroxyl content (15.48–19.04 mmol/g) make these microspheres suitable for protein adsorption. Meanwhile, bovine serum albumin was used to examine the adsorption capacity of the microspheres. These microspheres had a capacity as high as 49.28 mg/g. The adsorption kinetics and recycling of the beads were also investigated. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 5934–5940, 2006