Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.

A method is presented for locating protein antigenic determinants by analyzing amino acid sequences in order to find the point of greatest local hydrophilicity. This is accomplished by assigning each amino acid a numerical value (hydrophilicity value) and then repetitively averaging these values along the peptide chain. The point of highest local average hydrophilicity is invariably located in, or immediately adjacent to, an antigenic determinant. It was found that the prediction success rate depended on averaging group length, with hexapeptide averages yielding optimal results. The method was developed using 12 proteins for which extensive immunochemical analysis has been carried out and subsequently was used to predict antigenic determinants for the following proteins: hepatitis B surface antigen, influenza hemagglutinins, fowl plague virus hemagglutinin, human histocompatibility antigen HLA-B7, human interferons, Escherichia coli and cholera enterotoxins, ragweed allergens Ra3 and Ra5, and streptococcal M protein. The hepatitis B surface antigen sequence was synthesized by chemical means and was shown to have antigenic activity by radioimmunoassay.

https://www.pnas.org/content/pnas/78/6/3824.full.pdf

Prediction of protein antigenic determinants from amino acid sequences.

Solubilization of membranes by detergents

Analysis of membrane and surface protein sequences with the hydrophobic moment plot.

https://www.cell.com/cell/pdf/0092-8674(87)90086-9.pdf

A C-terminal signal prevents secretion of luminal ER proteins

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

The genes coding for the three membrane polypeptides of Semliki Forest virus have been sequenced and the primary structures of the proteins deduced. The amino acid sequence gives further insight into how the transmembrane structure of the three-chain virus membrane glycoprotein is generated in the infected cell.

Nucleotide sequence of cDNA coding for Semliki Forest virus membrane glycoproteins

In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

Semliki Forest virus expression system: production of conditionally infectious recombinant particles.

The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.

Henrik Garoff

Papers

A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

Nucleotide sequence of cDNA coding for Semliki Forest virus membrane glycoproteins

Semliki Forest virus expression system: production of conditionally infectious recombinant particles.

Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and β2-microglobulin