Co-expression of plastid chaperonin genes and a synthetic plant Rubisco operon in Escherichia coli

It has been suggested that lack of specialized molecular chaperone function(s) in Escherichia coli may account for the fact that although E. coli cells transformed with plant Rubisco genes synthesize the Rubisco subunit polypeptides, the active enzyme fails to assemble. If so, co-expression of plant chaperone and Rubisco genes might permit plant Rubisco assembly in E. coli. Introduction of genes encoding plant chaperonin polypeptides has been shown to enhance the capacity of E. coli to assemble active cyanobacterial Rubisco. We now report that co-expression of plant Rubisco and chaperonin genes affected the solubility and stability of Rubisco large subunit polypeptides, however, neither the assembled oligomeric protein nor Rubisco enzyme activity was detected.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Plant nuclear tRNAMet genes are ubiquitously interrupted by introns

We have isolated three independent clones for nuclear elongator tRNA^Met genes from an Arabidopsis DNA library using a tRNA^Met-specific probe generated by PCR. Each of the coding sequences for tRNA^Met in these clones is identical and is interrupted by an identical 11 bp long intervening sequence at the same position in the anticodon loop of the tRNA. Their sequences differ at two positions from the intron in a soybean counterpart. Southern analysis of Arabidopsis DNA demonstrates that a gene family coding for tRNA^Met is dispersed at at least eight loci in the genome. The unspliced precursor tRNA^Met intermediate was detected by RNA analysis using an oligonucleotide probe complementary to the putative intron sequence. In order to know whether introns commonly interrupt plant tRNA^Met genes, their coding sequences were PCR-amplified from the DNAs of eight phylogenetically separate plant species. All 53 sequences determined contain 10 to 13 bp long intervening sequences, always positioned one base downstream from the anticodon. They can all be potentially folded into the secondary structure characteristic for plant intron-containing precursor tRNAs. Surprisingly, GC residues are always present at the 5-distal end of each intron.     

Two streptokinase genes are expressed with different solubility in Escherichia coli W3110

The streptokinase (SK) gene from S. equisimilis H46A (ATCC 12449) was cloned in E. coli W3110 under the control of the tryptophan promoter. The recombinant SK, which represented 15% of total cell protein content, was found in the soluble fraction of disrupted cells. The solubility of this SK notably differed from that of the product of the SK gene from S. equisimilis (ATCC 9542) which had been cloned in E. coli W3110 by using similar expression vector and cell growth conditions, and occurred in the form of inclusion bodies.     

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 30% of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gp11, before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 DNA have 0.1% and mini cells have 1% of the gp11 levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gp10 and gp11 formed from the cloned genes after T4 infection.     

Expression of Chlamydia trachomatis inclusion membrane protein genes IncB and IncC in Escherichia coli

In this study, we have cloned the Chlamydia trachomatis genes incB and incC into the expression plasmid vectors from pET series for the subsequent isolation of recombinant proteins. As a result, we have obtained the first full-length recombinant C. trachomatis proteins IncB and IncC, which can be used for following antibody production and for study of their protein-protein interaction.     

利用植物生产异源蛋白的研究进展

植物作为生产异源蛋白的生物反应器,近年来颇受关注,与复杂,昂贵的以细胞培养为基础的表达系统相比,具有安全、廉价及规模化生等特点。作者简述了利用植物生产外源蛋白的主要策略及异源基因在植物中表达的研究进展。     

Efficient Expression of the Paramecium Calmodulin Gene in Escherichia coli after Four TAA-to-CAA Changes through a Series of Polymerase Chain Reactions

We have expressed the Paramecium calmodulin gene in Escherichia coli by changing the four TAA codons in this gene to CAAs. This was carried out by three polymerase chain reactions (PCRs) and then cloning the product into the expression vector pKK223-3 immediately downstream of its trp-lac hybrid promoter. JM109 strain of E. coli , transformed with the recombinant plasmid harboring the altered Paramecium calmodulin gene, produces a protein judged to be calmodulin. It is recognized by a monoclonal antibody to Paramecium calmodulin; it migrates with the native protein at nearly the same rate in electrophoreses; and it shows a Ca²⁺-dependent shift in electrophoretic pattern. The production of calmodulin is about 170 times as efficient with E. coli as with Paramecium in terms of unit volume of packed cells, and is about 400 times as efficient in unit volume of liquid culture. This method appears useful in site-directed mutageneses and in the heterologous productions of other ciliate proteins. A critique of this method is provided. A calmodulin half-molecule, a by-product of this project, is described.     

Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes

Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

sucAB and sucCD are mutually essential genes in Escherichia coli

sucAB and sucCD of Escherichia coli encode enzymes that generate succinyl-CoA from 2-oxoglutarate and succinate, respectively. Their mutual essentiality was studied. sucAB and sucCD could be deleted individually, but not simultaneously. The mutual essentiality of sucAB and sucCD was further confirmed by the conditional expression of sucABCD, sucAB, and sucCD under the control of a P(BAD) in E. coli MG1655, E. coli MG1655 (DeltasucCD), and E. coli MG1655 (DeltasucAB), respectively. These strains grew well in Luria-Bertani medium containing 0.1% arabinose, but not in the absence of arabinose unless the medium was supplemented with succinyl-CoA. Our results indicate that either sucAB or sucCD is enough to produce succinyl-CoA that is essential for cell viability.