Translation of bacteriophage T4 mRNA into active lysozyme by a wheat germ cell-free system.

T4 bacteriophage mRNA for lysozyme was extracted from T4 phage infected $\text{E. coli}$ cells, partially purified by column chromatography, and translated in a heterologous cell-free protein synthesizing system prepared from wheat germ. The translation product was confirmed by SDS polyacrylamide gel electrophoresis and enzymatic activity — bacteriolysis as tested with $\text{Micrococcus luteus}$. The specific activity of the enzyme prepared was 660 U/mg.     

Effects of 6-thioguanine-containing RNA on in vitro protein synthesis

6-Thioguanine was administered to rats 12 hr after partial hepatectomy at a dose of 40 mg/kg of body weight; 6 hr later, polyadenylic acid-containing RNA was isolated and was used to measure initiation of protein synthesis $\text{in vitro}$ in a wheat germ system. $\text{In vitro}$ initiation was found to be 2.3-fold greater when 6-thioguanine-containing RNA was employed, than when polyadenylic acid-containing RNA isolated from untreated animals was used. The homopolymer, poly(TG), did not promote peptide synthesis in the wheat germ $\text{in vitro}$ system employed.     

Identification of the covalently bound flavin of L-gulono-gamma-lactone oxidase.

A specific mRNA for a structural lipoprotein of the $\text{Escherichia}\text{coli}$ outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.     

Prolonged lifetime of the 410-nm intermediate of bacteriorhodopsin in the presence of guanidine hydrochloride.

Prostatic binding protein (PBP) is a quantitatively important steroid-binding protein present in rat ventral prostate. Electrophoresis on SDS-containing polyacrylamide gels shows that PBP is composed of two subunits, F and S having molecular weights of 16,000 and 18,000. Upon reduction these subunits dissociate further into smaller components. Translation of mRNA from rat ventral prostate in a wheat germ cell-free system or in $\text{Xenopus}$ oocytes results in the formation of polypeptides immunoprecipitable with an anti-PBP antiserum. However, as opposed to the wheat germ system, only the oocytes synthesize polypeptides, that are electrophoretically identical to those of native cytosolic PBP.     

In vitro synthesis of adrenodoxin and adrenodoxin reductase: Existence of a putative large precursor form of adrenodoxin

Cytoplasmic free and bound polysomes were isolated from bovine adrenal cortex, and used to program $\text{in}\text{vitro}$ protein synthesis in rat liver cell sap and wheat germ lysate systems. Synthesis of adrenodoxin(Ad) and adrenodoxin reductase(AdR) in the cell-free systems was determined by immunoprecipitation using monospecific antibodies, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Ad was synthesized by both free and bound polysomes as a putative large precursor having molecular weight of approximately 20,000 daltons, which was processed to mature size Ad (MW 12,000 daltons) by $\text{in}\text{vitro}$ incubation with adrenal cortex mitochondria. On the other hand, AdR was synthesized only by free polysomes apparently as the mature size product.     

Enhancing effect of magnesium ion on cell-free synthesis of read-through protein of bacteriophage Qbeta.

This report describes the enhancing effect of magnesium ion on the synthesis of read-through protein of bacteriophage Qβ in a cell-free protein synthesizing system from $\text{E. coli}$. At 6 mM of magnesium acetate, the major product was coat protein. At 12 mM of magnesium, it was replaced by read-through protein. This enhanced synthesis was substituted by the addition of 0.25 mM of spermine or 1 mM of spermidine to 6 mM of magnesium. These results suggest that magnesium or combination of magnesium and polyamines causes leaky termination at the end of the coat protein cistron of Qβ-RNA.     

Cell-free synthesis of angler fish preproinsulin: Complete amino acid sequence of the signal peptide

Messenger ribonucleic acid isolated from angler fish ($\text{Lophius}\text{americanus}$) islets of Langerhans was translated in the wheat germ cell-free protein synthesizing system containing different combinations of radioactive amino acids. Preproinsulin (～ 11,000 daltons) was identified amongst the translation products, by sodium dodecyl sulfate gel electrophoresis, and subjected to microsequencing techniques. The fish preproinsulin was found to possess an NH₂-terminal signal peptide of 24 amino acids, with regions of homology to human, rat and chicken preproinsulin signal sequences.     

Cryptic form of mRNA in dormant Artemia salina cysts.

Dormant $\text{Artemia salina}$ cysts are almost devoid of polysomal structures but contain appreciable quantities of mRNA that sediments mainly as a 40S complex in sucrose gradients. The mRNA can be isolated from this complex and efficiently translated in a wheat germ cell-free system, although the 40S complex itself is inactive. During rehydration of the cysts, mRNA becomes increasingly involved in polysomal complexes which can be actively translated in the cell-free system.     

In vitro synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β) and microsomal cytochrome P-450(C-21) by both free and bound polysomes isolated from bovine adrenal cortex

$\text{In}\text{vitro}$ synthesis of mitochondrial cytochromes P-450(scc) and P-450(11-β), and microsomal cytochrome P-450(C-21) programmed by bovine adrenal cortex polysomes was carried out using rat liver cell sap and wheat germ lysate systems. Synthesis of P-450 proteins in the cell-free systems was determined by immunoprecipitation and immunoadsorption using mono-specific antibodies to each species of P-450, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Both free and bound polysomes synthesized these three species of P-450 in the cell-free systems. P-450(scc) and P-450(C-21) were synthesized apparently as the mature size products, whereas P-450(11-β) was synthesized as a putative precursor approximately 5,000 daltons larger than the mature form. Mitochondrial and microsomal P-450 proteins seem to share common sites of synthesis in the cytoplasm of adrenal cortex cells.     

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Lactate dehydrogenase-C (LDH-C) mRNA was purified from $\text{DBA}\text{2}$ mouse testes and translated $\text{in}\text{vitro}$. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The acid-base properties and kinetics of dissolution of the Fe4S4 cores of Chromatin ferredoxin and high potential iron protein.

From a study of the translation of synthetic polynucleotides in the $\text{E. coli}$ and $\text{B. thuringiensis}$ cell-free systems, it is shown that the stimulation of polypeptide synthesis by spermidine depends on the uracil content of messenger ribonucleic acid. This stimulation can not be fulfilled by any amount of Mg²⁺ in the absence of polyamines.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Hormonal regulation of epidermis-specific protein and messenger RNA synthesis in amphibian metamorphosis

Experiments using a monospecific antibody directed against one type of epidermis-specific keratin from adult skin of the amphibian Xenopus laevis have demonstrated that polysomes synthesizing this protein first appear within larval skin during natural metamorphosis. Further experiments demonstrated that the synthesis of keratin within larval skin could be induced precociously by the thyroid hormone, 3,3′,5-triiodo-l-thyronine, both in vivo and when the isolated larval skin is cultured in vitro. The earliest developmental age responsive to such hormone induction appeared to be Stage $\text{50}\text{52}$ of larval development. This is about 20–24 days before keratin would normally make its appearance within the skin during natural metamorphosis. Hormone treatment of tadpoles at this age will also cause a precocious increase in the amount of keratin messenger RNA present within larval skin. This has been demonstrated directly by the isolation of poly(A)-containing messenger RNA from hormone-treated larvae and its translation in a wheat germ cell-free system to give immunoprecipitable keratin. Peptide analysis of the in vitro translation product indicates that the hormone-induced mRNA probably codes for an initial protein product that is slightly larger than keratin itself.     

Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability

Creatine phosphokinase is used to generate ATP with creatine phosphate for $\text{in vitro}$ protein synthesis. Some preparations of this enzyme contain nuclease activity, which can be demonstrated by a sensitive assay of the cleavage of poly(A)-containing RNA. These preparations of creatine phosphokinase support protein synthesis poorly in a cell-free system prepared from HeLa cells. Poly(A)-containing RNA is quite stable in this cell-free system when the phosphorylated sugar fructose 1,6-bisphosphate with no addition of enzyme is used to generate ATP.     

The effect of dietary fat on the activity,content, rates of synthesis,and degradation and translation of messenger RNA coding for malic enzyme in mouse liver

The specific activity (units activity/mg cytosolic protein) of malic enzyme was found to be three-fold higher in the livers of mice fed a semipurified diet containing 50% ($\text{w}\text{w}$) glucose and 15% ($\text{w}\text{w}$) saturated and monounsaturated but no polyunsaturated fat (hydrogenated cottonseed oil) over an 11-day period than in the livers of mice fed a standard laboratory mouse chow (Purina) diet. In contrast, when other lab chow-fed mice were fed an isocaloric diet containing 15% ($\text{w}\text{w}$) polyunsaturated fat (corn oil), no change in the specific activity of malic enzyme occurred over a similar period of time. Rocket immunoelectrophoresis performed on cytosols from both dietary groups demonstrated that the livers of mice consuming the hydrogenated cottonseed oil diet contained approximately three times more malic enzyme protein than did the livers from the corn oil-fed animals. In mice pulse-labeled with l-[4,5-³H]leucine, the rate of hepatic malic enzyme synthesis (relative to that for total protein) was approximately twofold greater in the hydrogenated cottonseed oil-fed mice than in their corn oil-fed counterparts whereas the rate of hepatic malic enzyme degradation was similar for both groups. Immunotitration of liver malic enzyme from hydrogenated cottonseed oil-fed and corn oil-fed mice revealed identical equivalence points, demonstrating that the catalytic efficiency of mouse liver malic enzyme had not been affected by the type of dietary fat administered. When total liver RNA, isolated from the hydrogenated cottonseed oil- and the corn oil-fed animals, was translated in cell-free translation systems (wheat germ extract and reticulocyte lysate) we found that both dietary treatments had resulted in an increase in the activity of malic enzyme messenger RNA. Furthermore, there were no significant differences between the two dietary groups in this regard. These results suggest that hepatic malic enzyme specific activity in high-carbohydrate polyunsaturated fat-fed mice is regulated principally by dietary-induced changes in the rate of enzyme synthesis and not by the activity of messenger RNA coding for the enzyme.     

Translation of AKR-murine leukemia viral RNA in an E. coli cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an $\text{E. coli}$ cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized $\text{in vitro}$ in response to AKR-MuLV RNA in the $\text{E. coli}$ cell-free system.     

Cell-free translation of messenger RNAs of embryonic tooth organs: Synthesis of the major extracellular matrix proteins

In order to understand the regulation of embryonic mammalian enamel and dentine extracellular matrix protein synthesis, the biological activity of embryonic rabbit molar tooth organ messenger RNAs has been examined. Total RNA was extracted from 26-day embryonic tooth organs and fractionated by chromatography on oligo(dT)-cellulose. Replicate samples were fractionated on sucrose density gradients and the poly(A)-containing distribution determined using a poly(U) ${}^3$H assay. The poly(A)-containing fractions stimulated ${}^3$H-proline incorporation 10-fold in wheat germ cell-free extracts. Analysis of the labelled reaction products on sodium dodecyl sulphate-polyacrylamide gels revealed seven major peaks, one co-migrating with procollagen alpha chains (circa 145,000 daltons) and the others migrating slightly faster than the various extracellular matrix proteins which characterize amelogenesis and dentinogenesis. Purified collagenase digestion of the cell-free reaction products eliminated the 145,000 dalton procollagen-like polypeptide. This is the first demonstration of the isolation of embryonic tooth organ messenger RNAs and provides an experimental approach by which to study the regulation of extracellular matrix formation during tooth morphogenesis. We predict that the non-collagenous proteins synthesized $\text{in vitro}$ represent enamel proteins, alkaline phosphatase, dentine phosphoproteins and proteins associated with proteoglycans.     

Effect of polyamines on the fidelity of macromolecular synthesis

Addition of polyamines to in vitro systems containing suboptimal concentrations of Mg2+ markedly stimulated protein and RNA synthesis. This stimulation is observed only within a marrow range of polyamine concentration. The extend of stimulation of RNA synthesis was dependent on assay conditions. Addition of spermidine to the wheat germ system not only stimulated poly(U) directed polyphenylalanine synthesis, but also reduced the misincorporation of leucine. MS2-coat protein synthesis, studied in an E.coli cell-free system using either one of the two glutamyl-tRNAs as the only source of glutamine, suggested that in the presence of spermidine, codon recognition by these two isoacceptor tRNA molecules was more stringent. From these results it is concluded that polyamines contribute to the specificity of codon/anticodon interactions and thereby increase the fidelity of protein synthesis.     

Modeccin — A plant toxin inhibiting protein synthesis

Modeccin is shown to strongly inhibit the ability of HeLa cells to form colonies $\text{in vitro}$. In modeccin treated cells the rate of incorporation of labelled leucine was reduced earlier than that of uridine and thymidine, and the toxin also inhibited protein synthesis in a cell-free system from rabbit reticulocytes. When modeccin was added to human erythrocytes agglutination was induced upon subsequent addition of anti-modeccin indicating that the toxin binds to cell surfaces. This effect was strongly increased after neuraminidase treatment of the cells. Furthermore, neuraminidase treatment of HeLa cells strongly increased their sensitivity to modeccin. The data indicate that modeccin acts by binding to cell surfaces and then somehow interferes with protein synthesis.     

Wheat germ systems for cell-free protein expression

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.