Ibrahim M. Ibrahimi

A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo.

Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.

A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription‐coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine‐labelled proteins produced offer a number of possibilities for the study of structure‐function relationships of proteins.

Evolutionary substitutions and the antigenic structure of globular proteins

A FEW years ago, Crumpton1 and Reichlin2 reviewed information about the antigenic structure of globular proteins of known amino acid sequence and three-dimensional structure. It seemed that only a small fraction of the amino acid residues in such proteins participated directly in antibody binding. This impression was reinforced by Atassi and co-workers, who proposed antigenic structures of two such proteins—sperm whale myoglobin3 and chicken lysozyme c (ref.4). These workers claim to have identified all those amino acid residues which bind to antibodies present in antisera directed against the protein. These claims stem from extensive studies with chemically modified forms of the protein as well as with antigenically active fragments obtained by specific cleavage of the protein. The antigenic residues are arranged in groups called determinants, each of which binds a specific class of antibodies. A typical determinant contains four to seven residues. For both lysozyme and myoglobin, the proportion of antigenic residues is about 15% of the total number of amino acid residues in the protein3,4. Information has also accumulated concerning the power of antisera to detect evolutionary substitutions of amino acids in globular proteins. Quantitative immunological comparisons of related proteins of known amino acid sequence have consistently shown a strong correlation between degree of sequence difference and degree of antigenic difference. This has been demonstrated most thoroughly for monomeric globular proteins such as lysozymes5,6, ribonucleases7, azurins8 and cytochromes c (refs 9, 10). Less complete information suggests that the correlation holds also for plastocyanins10, tryptophan synthetase α subunits11, serum albumins12, carbonic anhydrases and myoglobins13 (A. B. Champion, E. M. Prager, S. L. Welch, and A. C. Wilson, unpublished), and ferredoxins14. From the strength of the correlations observed for lysozymes, ribonucleases, azurins and cytochromes (r > 0.9), one may make the statistical inference that about 80% (that is, 0.9 squared) of those amino acid substitutions which accumulate during evolution are immunologically detectable7. Here we summarise additional evidence that the majority of evolutionary substitutions are immunologically detectable, and we discuss how to reconcile this evidence with the chemical evidence that only a small fraction of the amino acids are antigenic.

The effect of a single amino acid substitution on the antigenic specificity of the loop region of lysozyme

Abstract Immunological studies were carried out with four closely related lysozymes—those of chicken, California quail, bobwhite quail, and chukar partridge—using anti-lysozyme and anti-loop antibodies. With micro-complement fixation and phage inhibition assays, it was shown that substitution of lysine for arginine at position 68 influences the binding of antibodies to lysozyme. Furthermore, the magnitude of the antigenic differences among the four lysozymes correlated with the nature of the amino acid at that position. The partial amino acid sequence of partridge lysozyme is presented in this paper. The partridge lysozyme sequence data were valuable for interpretation and quantitation of the immunochemical differences among the other three lysozymes because at some positions in the sequence the partridge sequence was chicken-like and at other positions it was California quail-like.

Electrophoretic polymorphism in rabbit tear lysozyme

Rabbit tears were found to contain two lysozymes which differed in their electrophoretic mobility and were designated tear lysozymes 1 and 2. Rabbit tear lysozyme 1 was purified to homogeneity by conventional purification methods. It was found to be distinct from other known mammalian c-type lysozymes, rabbit tear lysozyme 2 and the major rabbit gastrointestinal lysozyme. The activity profile is centered around the neutral region with an optimum of 7 which is slightly lower than that for chicken lysozyme. The thermal stability as well as inhibition profiles by the substrate analogues, N-acetylglucosamine (NAG) and chitotetraose (NAG)4 are comparable to those of chicken lysozyme. Based on its molecular weight and catalytic properties this isozyme is classified as a c-type lysozyme.