A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Bacteriophage lambda (λ) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current λ display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient λ lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage λ and to facilitate the use of modified λ phage vectors in mammalian gene transfer applications.     

A simple method for constructing a tagged protein

We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.     

A simple probabilistic scoring method for protein domain identification

SUMMARY: A simple heuristic scoring method is described for assigning sequences to known domain types based on BLAST search outputs. The scoring is based on the score distribution of the known domain groups determined from a database versus database comparison and is directly applicable to BLAST output processing.     

A Pascal microcomputer program for prediction of protein secondary structure and hydropathic segments

This paper describes a simple Pascal microcomputer program forprediction of protein secondary structure according to the Chouand Fasman algorithm. In addition, it performs an analysis ofthe hydropathic character of the residues for prediction ofexternal/internal regions of the polypeptide chain. Also itsearches for probable glycosylation and phosphorylation sites. Received on July 26, 1985; accepted on May 6, 1986     

A method for the induction of blood eosinophilia with simple protein antigens

A new method of immunization is presented in which blood eosinophilia is induced with simple protein antigens in rats. Large inert particles coated with antigen are injected intravenously embolizing the lungs. Eosinophilia is maximal 5 days after a booster injection. It is concluded that the site of tissue localization of antigen is important in blood eosinophilia induction.     

A simple and rapid method for isolating myelin basic protein

The conduction of impulses along axons of nerves is facilitated by the myelin sheath, composed of proteins and lipid. Myelin basic proteins (MBPs) are extrinsic membrane proteins that play an important role in the structural organization of the myelin sheath. In the central nervous system, MBPs account for 30-40% of total protein. The traditional method of MBP isolation involves the use of chloroform-ethanol, which would destroy the native form of MBP. A modified method for maintaining its native form was developed. The white matter of porcine brain was directly extracted by buffers containing different concentrations of sodium chloride owing to MBP solubilized at high concentration of NaCl. The MBP was further purified by cation exchange chromatography and buffers containing glycine and salts. Purified MBP were consistently obtained by this method.     

A simple method for detecting protein antigens in flour.

Wheat, barley, rye or oat flour was dissolved in 0.2N NaOH using magnetic stirring followed by sonication. The ensuing clear solutions contained 95% of protein in the dry matter. Aliquots were electrophoresed on 12% polyacrylamide gels which were either stained with Coomassie brilliant blue or Western-blotted on nitrocellulose membrane. Incubation of the latter with serum from persons sensitized to flours allowed the detection of antigenic flour protein classes. It seems that many antigens are present in common kitchen flour.     

A rapid and simple method for staining of the crystal protein ofBacillus thuringiensis

Summary A rapid and simple method of staining for the crystal protein (-endotoxin or parasporal body) ofBacillus thuringiensis has been developed. Changes in colonial morphology were observed when cells lost their ability to form crystal protein or both crystal protein and spore.     

A simple fluorescent method for quantitative determination of aortic protein uptake.

A quantitative system for direct protein tracing and measurement of net protein uptake in the aorta using fluorescein isothiocyanate conjugated bovine serum albumin (FITCBSA) is described. Using Wistar rats, a mean aortic FITCBSA net uptake of 29.7 times 10(-17) g FITCBSA per mum2 aortic endothelial surface area per 24 h was obtained. Intra-aortic localization of the FITCBSA was observed along the endothelium and the collagen-elastin bands. The values obtained using this FITCBSA system are comparable with those of a previously established isotopic technique measuring aortic albumin flux and reconfirm the previous findings of the existence of an albumin permeability gradient in the thoracic aorta.     

A simple rapid biuret method for the estimation of protein in samples containing thiols

Interference in the Lowry protein determination by thiol compounds is now well known (1–3). We have found that the estimation of protein by the biuret reaction is also subject to interference when the protein sample contains various thiols. We wish to report that this interference can be prevented in most cases by using a biuret reagent which is chelated with ethylenediaminetetraacetate (EDTA). In samples containing dithiothreitol (DTT) it is also necessary to add iodoacetamide prior to the addition of the biuret reagent. The use of iodoacetate to eliminate thiol interference in the Lowry procedure has been reported previously (3).This report details the extent of interference of dithiothreitol, β-mercaptoethanol, β-mercaptoethylamine, and glutathione, and illustrates the extent of neutralization which is attained in each case. We have also introduced modifications which permit the development of a stable color in only 5 min.     

SCOOP: a simple method for identification of novel protein superfamily relationships

MOTIVATION: Profile searches of sequence databases are a sensitive way to detect sequence relationships. Sophisticated profile-profile comparison algorithms that have been recently introduced increase search sensitivity even further. RESULTS: In this article, a simpler approach than profile-profile comparison is presented that has a comparable performance to state-of-the-art tools such as COMPASS, HHsearch and PRC. This approach is called SCOOP (Simple Comparison Of Outputs Program), and is shown to find known relationships between families in the Pfam database as well as detect novel distant relationships between families. Several novel discoveries are presented including the discovery that a domain of unknown function (DUF283) found in Dicer proteins is related to double-stranded RNA-binding domains. AVAILABILITY: SCOOP is freely available under a GNU GPL license from http://www.sanger.ac.uk/Users/agb/SCOOP/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

A method for estimating the effective number of loci affecting a quantitative character

A likelihood method is introduced that jointly estimates the number of loci and the additive effect of alleles that account for the genetic variance of a normally distributed quantitative character in a randomly mating population. The method assumes that measurements of the character are available from one or both parents and an arbitrary number of full siblings. The method uses the fact, first recognized by Karl Pearson in 1904, that the variance of a character among offspring depends on both the parental phenotypes and on the number of loci. Simulations show that the method performs well provided that data from a sufficient number of families (on the order of thousands) are available. This method assumes that the loci are in Hardy–Weinberg and linkage equilibrium but does not assume anything about the linkage relationships. It performs equally well if all loci are on the same non-recombining chromosome provided they are in linkage equilibrium. The method can be adapted to take account of loci already identified as being associated with the character of interest. In that case, the method estimates the number of loci not already known to affect the character. The method applied to measurements of crown–rump length in 281 family trios in a captive colony of African green monkeys (Chlorocebus aethiopus sabaeus) estimates the number of loci to be 112 and the additive effect to be 0.26 cm. A parametric bootstrap analysis shows that a rough confidence interval has a lower bound of 14 loci.     

POCKET: A computer graphies method for identifying and displaying protein cavities and their surrounding amino acids

A new interactive graphics program is described that provides a quick and simple procedure for identifying, displaying, and manipulating the indentations, cavities, or holes in a known protein structure. These regions are defined as, e.g., the X₀, y₀, Z₀ values at which a test sphere of radius r can be placed without touching the centers of any protein atoms, subject to the condition that there is some x < x₀ and some x > x₀ where the sphere does touch the protein atoms. The surfaces of these pockets are modeled using a modification of the marching cubes algorithm. This modification provides identification of each closed surface so that by “clicking” on any line of the surface, the entire surface can be selected. The surface can be displayed either as a line grid or as a solid surface. After the desired “pocket” has been selected, the amino acid residues and atoms that surround this pocket can be selected and displayed. The protein database that is input can have more than one protein “segment,” allowing identification of the pockets at the interface between proteins. The use of the program is illustrated with several specific examples. The program is written in C and requires Silicon Graphics graphics routines.     

A method of displaying differences in botanical composition

A new graphical method for displaying interrelationships within data on botanical composition of communities is described. It employs plotting roses of vectors where direction indicates species and length, proportional representation. The use and advantages of the method are discussed.     

A simple method for the purification of myxobacteria

Pure cultures of many myxobacteria could be quickly obtained by treating fruiting bodies with highly dosed mixtures of antibiotics