The role of the transit peptide in the routing of precursors toward different chloroplast compartments

A new approach for adenosine triphosphate (ATP) regeneration in a cell-free protein synthesis system is described. We first show that pyruvate can be used as a secondary energy source to replace or supplement the conventional secondary energy source, phosphoenol pyruvate (PEP). We also report that glucose-6-phosphate, an earlier intermediate of the glycolytic pathway, can be used for ATP regeneration. These new methods provide more stable maintenance of ATP concentration during protein synthesis. Because pyruvate and glucose-6-phosphate are the first and last intermediates of the glycolytic pathway, respectively, the results also suggest the possibility of using any glycolytic intermediate, or even glucose, for ATP regeneration in a cell-free protein synthesis system. As a result, the methods described provide cell-free protein synthesis with greater flexibility and cost efficiency.

Regeneration of adenosine triphosphate from glycolytic intermediates for cell‐free protein synthesis

Cell-free expression is emerging as a prime method for the rapid production of preparative quantities of high-quality membrane protein samples The technology facilitates easy access to large numbers of proteins that have been extremely difficult to obtain Most frequently used are cell-free systems based on extracts of Escherichia coli cells, and the reaction procedures are reliable and efficient This protocol describes the preparation of all essential reaction components such as the E coli cell extract, T7 RNA polymerase, DNA templates as well as the individual stock solutions The setups of expression reactions in analytical and preparative scales, including a variety of reaction designs, are illustrated We provide detailed reaction schemes that allow the preparation of milligram amounts of functionally folded membrane proteins of prokaryotic and eukaryotic origin in less than 24 h

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC 3.2.1.15] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)+ RNA isolated from ripe tomato fruit. The cDNA library was inserted into a λ-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

https://www.pnas.org/content/pnas/83/17/6420.full.pdf

Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening.

Mixtures containing a biological macromolecule, such as a nucleic acid molecule or a polypeptide, and a liquid matrix, which absorbs infrared (IR) radiation, are provided. These mixtures are useful for analysis of the biological macromolecule by IR matrix assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Also provided are processes for analyzing a biological macromolecule using IR-MALDI mass spectrometry. For example, processes for detecting the presence or identity of a biological macromolecule in a sample, or for sequencing a biological macromolecule are provided.

Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules

Publisher Summary This chapter describes the preparation of a cell-free protein-synthesizing system from wheat germ. Crude extracts of commercial wheat germ are known to be capable of translating a wide variety of messenger RNAs. Most wheat germ extracts have low endogenous activity. If necessary, endogenous activity may be further reduced by a preincubation step with creatine phosphokinase or by treatment with micrococcal nuclease. Even with extracts that have a relatively high endogenous activity as determined by the incorporation of radioactive amino acids, few if any endogenous protein bands are observed on SDS-polyacrylamide gels. HEPES should be stored frozen in small aliquots; it supports microbial growth and it is not stable to autoclaving. One should also remember that the buffer supplies some potassium that has not been accounted for in the concentrations given.

Preparation of a cell-free protein-synthesizing system from wheat germ.

Publisher Summary
This chapter describes the preparation of a cell-free protein-synthesizing system from wheat germ. Crude extracts of commercial wheat germ are known to be capable of translating a wide variety of messenger RNAs. Most wheat germ extracts have low endogenous activity. If necessary, endogenous activity may be further reduced by a preincubation step with creatine phosphokinase or by treatment with micrococcal nuclease. Even with extracts that have a relatively high endogenous activity as determined by the incorporation of radioactive amino acids, few if any endogenous protein bands are observed on SDS-polyacrylamide gels. HEPES should be stored frozen in small aliquots; it supports microbial growth and it is not stable to autoclaving. One should also remember that the buffer supplies some potassium that has not been accounted for in the concentrations given.

38 – Preparation of a Cell-Free Protein-Synthesizing System from Wheat Germ

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Preparation of a cell-free protein-synthesizing system from wheat germ.

38 – Preparation of a Cell-Free Protein-Synthesizing System from Wheat Germ