Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels.

Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.

Human apolipoprotein E expression in Escherichia coli: structural and functional identity of the bacterially produced protein with plasma apolipoprotein E.

Human apolipoprotein E (apoE) was produced in Escherichia coli by transforming cells with an expression vector containing a reconstructed apoE cDNA, a lambda PL promoter regulated by the thermolabile cI repressor, and a ribosomal binding site derived from the lambda cII or the E. coli beta-lactamase gene. Transformed cells induced at 42 degrees C for short periods of time (less than 20 min) produced apoE, which accumulated in the cells at levels of approximately equal to 1% of the total soluble cellular protein. Longer induction periods resulted in cell lysis and the proteolytic destruction of apoE. The bacterially produced apoE was purified by heparin-Sepharose affinity chromatography, Sephacryl S-300 gel filtration, and preparative Immobiline isoelectric focusing. The final yield was approximately equal to 20% of the initial apoE present in the cells. Except for an additional methionine at the amino terminus, the bacterially produced apoE was indistinguishable from authentic human plasma apoE as determined by NaDodSO4 and isoelectric focusing gel electrophoresis, amino acid composition of the total protein as well as its cyanogen bromide fragments, and partial amino acid sequence analysis (residues 1-17 and 109-164). Both the bacterially produced and authentic plasma apoE bound similarly to apolipoprotein B,E(low density lipoprotein) receptors of human fibroblasts and to hepatic apoE receptors. Intravenous injection resulted in similar rates of clearance for both the bacterially produced and authentic apoE from rabbit and rat plasma (approximately equal to 50% removed in 20 min). The ability to synthesize a bacterially produced human apolipoprotein with biological properties indistinguishable from those of the native protein will allow the production of large quantities of apoE for use in further investigations of the biological and physiological properties of this apolipoprotein.

Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid.

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.

Nuclear RNA is spliced in the absence of poly(A) addition.

Splicing of newly formed nuclear RNA transcripts has been demonstrated during adenovirus type 2 (Ad2) mRNA formation in HeLa cells in the presence of "cordycepin," 3' deoxyadenosine, a drug that stops poly(A) addition to nuclear RNA. Nuclear RNA prepared from Ad2-infected cells after a 30 min label time in the presence or absence of 3' deoxyadenosine was hybridized to and eluted from Ad2 DNA sequences in the transcription units of region E1b and region E2. The nuclear RNA from the 3' dA-treated cells did not contain poly(A) but did contain Ad2-specific molecules approximately 200 to 250 bases shorter than the spliced mRNAs of the control infected cells. In addition, the approximately 2 kb RNA from Ad2 region E2 was shown to have sequences that lie more than 3.5 kb apart on the DNA, suggesting that correct cutting and splicing of the primary transcript in the absence of poly(A) synthesis had occurred. Therefore, although poly(A) addition usually precedes splicing during mRNA formation, poly(A) is not required for splicing.

Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies.

Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coli plasmid pBR322. The major fibroblast interferon (Hu IFN-beta 1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 Mr protein. The second interferon mRNA species (Hu IFN-beta 2) is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr. The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rI.rC), but IFN-beta 2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-beta 1 is not induced.

Identification of the translation products of human fibroblast interferon mRNA in reticulocyte lysates.

Messenger RNA was purified from human foreskin fibroblasts FS11, a high interferon-producer line, after induction with synthetic double-stranded RNA. The mRNA was translated in a cell-free protein-synthesis system from rabbit reticulocytes. The translation products, containing biologically active human interferon, were immunoprecipitated by a serum from rabbits immunized against partially purified interferon. Analysis of the immunoprecipitate by polyacrylamide gel electrophoresis in dodecylsulfate shows that the product of human fibroblast interferon mRNA is a 23000-Mr polypeptide. Methods are described for the synthesis and rapid identification of this polypeptide, which should be useful for structural analysis of interferon and isolation of its mRNA.

A streptomycin-resistant Escherichia coli mutant with ribosomes temperature-sensitive in the suppression of a nonsense codon.

Cell free extracts from a streptomycin-resistant E. coli mutant which is also temperature-sensitive for Q beta phage were studied for suppression of a nonsense mutation at various temperatures. The streptomycin-resistant ribosomes of the mutant were found to be temperature-sensitive in suppression of an amber mutation in f2 phage coat protein while retaining the ability to synthesize proteins at an elevated temperature (42 degrees C). The restriction of amber suppression at 42 degrees C is assumed to be related to an alteration in ribosomal protein S12 of the streptomycin-resistant mutant which also causes a change in its electrophoretic mobility.

Preferential charging of tRNA-Met-f in Escherichia coli K12.

The charging of tRNA-Met-f and tRNA-Met-m in vivo and in vitro and initiation of polysomes during methionine limitation were studied in two strains of Escherichia coli K12. In the wild-type strain the distribution of polysomes as well as the kinetic parameters of methionyl-tRNA synthetase indicate preferential acylation of tRNA-Met-f. This preferential charging of tRNAM-et-f does not take place in a mutant strain which is also defective in initiation of polysomes during methionine limitation.

In vitro synthesis of tRNA precursors and their conversion to mature size tRNA.

Two Escherichia coli tRNA gene clusters, tRNA1Tyr (su3+ and su3-) and tRNA2Tyr, tRNA2Gly (su+36), tRNA3Thr, were transcribed in a purified in vitro system. Evidence indicates that the adjacent tRNA genes are transcribed together as a common precursor of large size, which, on incubation with crude cell extracts, yields mature tRNA molecules.