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171.
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173.
Sun Ying-Chieh; Veenstra David L.; Kollman Peter A. 《Protein engineering, design & selection : PEDS》1996,9(3):273-281
Free energy calculations were carried out to determine the relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study. 相似文献
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175.
目的获得高质量的中国流行株HIV-1B/C重组亚型包膜蛋白抗原。方法改造表达载体,以构建中国流行株HIV-1B/C重组亚型包膜蛋白基因带有筛选标记的真核细胞表达质粒,将所构建质粒转染真核细胞,用含有抗性的培养基筛选出能够高效、持续表达包膜蛋白的稳定表达细胞株。结果所构建的表达载体pVRPEnv转染细胞后可表达目的蛋白,并建立了稳定表达细胞系。结论获得了可以高效、持续稳定表达中国流行株HIV-1B/C重组亚型包膜蛋白的细胞株。 相似文献
176.
Nielsen Per F.; Roepstorff Peter; Clausen Ib G.; Jensen Ejner B.; Jonassen Ib; Svendsen Allan; Balschmidt Per; Hansen Finn B. 《Protein engineering, design & selection : PEDS》1989,2(6):449-457
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. 相似文献
177.
Bates Paul A.; McGregor Malcolm J.; Islam Suhail A.; Sattentau Quentin J.; Sternberg Michael J.E. 《Protein engineering, design & selection : PEDS》1989,3(1):13-21
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. 相似文献
178.
Werten Paul J.L.; Carver John A.; Jaenicke Rainer; de Jong Wilfried W. 《Protein engineering, design & selection : PEDS》1996,9(11):1021-1028
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. 相似文献
179.
Blondel Arnaud; Nageotte Roland; Bedouelle Hugues 《Protein engineering, design & selection : PEDS》1996,9(2):231-238
Hybrid MalEGVP 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 MalEGVP. 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 MalEGVP 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. 相似文献
180.
Flaking and extrusion as mechanical treatments for enzyme-assisted aqueous extraction of oil from soybeans 总被引:6,自引:7,他引:6
B. P. Lamsal P. A. Murphy L. A. Johnson 《Journal of the American Oil Chemists' Society》2006,83(11):973-979
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. 相似文献