Production of a full length Tat protein in E. coli and its purification

A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We then defined a rapid, three-step procedure for the purification of the Tat protein.     

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.     

High-level gene expression for recombinant penicillin acylase production using the araB promoter system in Escherichia coli

The pac gene encoding penicillin acylase (PAC) was overexpressed under the regulation of the araB promoter (ParaB, also known as PBAD) in Escherichia coli (E. coli). The current ParaB expression system exhibited minimum leaking pac expression in the absence of arabinose as well as fast and high-level pac expression upon induction with arabinose in a wide concentration range. The production of PAC was limited by the accumulation of PAC precursors (i.e., proPAC in both soluble and insoluble forms) and various negative cellular responses, such as growth arrest and cell lysis. The culture performance could be improved by degP coexpression and the individual contribution of DegP protease and chaperone activities to the enhancement on the production of PAC was characterized. The study highlights the importance of identifying the step(s) limiting high-level gene expression and subsequent design and construction of the host/vector system for enhancing recombinant protein production in E. coli.     

In vivo regulation of the Escherichia coli araC promoter.

The ara pC promoter is known to be derepressed about fivefold for 20 to 30 min after the addition of arabinose. This transient derepression was studied by using araC::Mu lac insertions and araC-lacZ gene fusions. In strains containing increased levels of araC protein, the pC promoter became progressively less derepressible, but the ara pBAD promoter remained normally inducible. Repression of pC was reestablished 20 min after induction in araB mutants, but did not occur in arabinose-transport-deficient mutants. Finally, mutant araCc proteins which normally do not repress pC did so in the presence of arabinose.     

A new family of sugar-inducible expression vectors for Escherichia coli.

A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter. Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence. Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors. With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein.     

Polarity in gene araB of the l-Arabinose operon in Escherichia coli B/r

A series of mutations are described which map in the araB gene of the l-arabinose operon and exert a polar effect on gene araA, the structural gene for the l-arabinose isomerase. Ten of the 20 araB point mutants examined exhibited absolute polarity and may represent insertions of genetic material into the araB gene. The remaining 10 point mutants exhibit strong polarity (less than 10% of the normal wild-type inducible level of isomerase) and may represent a class of externally suppressible polar mutations other than amber or ochre. Seven of the 12 araB deletion mutants examined, or 58%, exhibit polarity, suggesting that a shift in the reading frame has been generated in the polycistronic message for the l-arabinose operon. The remaining, presumably in-phase, deletion mutants exhibit hyperinducible levels of isomerase, an effect that is eliminated when an araB(+) gene is introduced in the trans position. The hyperinducibility effect is discussed in terms of a model for self-catabolite repression, originally proposed by Katz and Englesberg.     

araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora.

The araB and araC genes of Erwinia carotovora were expressed in Escherichia coli and Salmonella typhimurium. The araB and araC genes in E. coli, E. carotovora, and S. typhimurium were transcribed in divergent directions. In E. carotovora, the araB and araC genes were separated by 3.5 kilobase pairs, whereas in E. coli and S. typhimurium they were separated by 147 base pairs. The nucleotide sequence of the E. carotovora araC gene was determined. The predicted sequence of AraC protein of E. carotovora was 18 and 29 amino acids longer than that of AraC protein of E. coli and S. typhimurium, respectively. The DNA sequence of the araC gene of E. carotovora was 58% homologous to that of E. coli and 59% homologous to that of S. typhimurium, with respect to the common region they share. The predicted amino acid sequence of AraC protein was 57% homologous to that of E. coli and 58% homologous to that of S. typhimurium. The 5' noncoding regions of the araB and araC genes of E. carotovora had little homology to either of the other two species.     

Inherited immunodeficiency with a defect in a major histocompatibility complex class II promoter-binding protein differs in the chromatin structure of the HLA-DRA gene.

A defect in a trans-regulatory factor which controls major histocompatibility complex class II gene expression is responsible for an inherited form of immunodeficiency with a lack of expression of human leukocyte antigen (HLA) class II antigens. We have recently described and cloned an HLA class II promoter DNA-binding protein, RF-X, present in normal B cells and absent in these class II-deficient regulatory mutants. Here we report that these in vitro results correlate with a specific change in the chromatin structure of the class II promoter: two prominent DNase I-hypersensitive sites were identified in the promoter of the HLA-DRA gene in normal B lymphocytes and found to be absent in the class II-deficient mutant cells. The same two prominent DNase I-hypersensitive sites were observed in normal fibroblastic cells induced by gamma interferon to express class II genes. Interestingly, they were also observed in the uninduced class II-negative fibroblastic cells, which have also been shown to have a normal RF-X binding pattern. We conclude that the two DNase I-hypersensitive sites in the HLA-DRA promoter reflect features in chromatin structure which correlate with the binding of the trans-acting factor RF-X and which are necessary but not sufficient for the expression of class II genes.     

Transfer vectors for maximal expression of passenger genes in the Bombyx mori nuclear polyhedrosis virus expression system

A series of Bombyx mori nuclear polyhedrosis virus (Bm-NPV) transfer vectors has been developed containing various lengths of the polyhedrin promoter, including sequences 3' of the initiation codon. The ATG initiation codon was mutated in some of these vectors to allow for the production of authentic nonfusion proteins. The ability of the various polyhedrin promoter constructs to direct expression of foreign gene sequences was assessed using two test genes, chloramphenicol acetyl transferase (cat), and human metallothionein II. Accumulation of cat mRNA and nonfused protein was low when only polyhedrin promoter sequences to -8 (relative to the translational start site of polyhedrin mRNA) were included in the transfer vector, but cat expression was comparable with that of the wild-type polyhedrin gene when promoter sequences to +5 were present. Further addition of polyhedrin gene sequences to +26 or +94 resulted in no further increase in expression. Similar results were obtained for expression of human metallothionein II, where constructs encoding polyhedrin-metallothionein fusion proteins containing polyhedrin sequences to at least +5 resulted in high levels of mRNA and protein accumulation. The expression vectors containing the +5, +26, or +94 BmNPV polyhedrin promoter can thus be used to direct maximal levels of production of nonfused proteins (when the polyedrin ATG has been mutated) or of fusion proteins, depending on which is more suitable for a particular application. These new vectors are a useful addition to those presently available and should increase the utility of the BmNPV expression system for large-scale protein production. (c) 1993 John Wiley & Sons, Inc.