Leonard E. Post

Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression

Sodium butyrate was used to enhance expression levels and thereby facilitate the generation of analytical quantities of nine different tissue plasminogen activator (tPA) analogues expressed under the control of the cytomegalovirus immediate early (CMV IE) promoter by the Chinese hamster ovary (CHO) mammalian expression system. Production involved growth in roller bottles, using serum free or low serum media formulations, together with repetitive, sodium butyrate inductions. Average inductions in the presence of sodium butyrate ranged from 2 to 9-fold relative to uninduced controls, using cell lines with no previous butyrate exposure. Retardation of growth rate by butyrate minimized the need to split cells during the production runs, extending longevity of roller bottles containing cells secreting at induced levels. SDS-PAGE analyses indicate a consistently high percentage of single-chain material. Measurements of specific activity and fibrinogen fragment enhancement for one of the analogues demonstrate that neither of these two critical parameters are affected by production in the presence of butyrate. Induction kinetic data and growth curves for the expression of sCD4 under control of the SV40 early promoter demonstrate that the benefits of butyrate can be realized with different promoters and heterologous genes, and are additive when used in conjunction with an amplified cell line constitutively expressing at elevated levels. This work demonstrates the practical application of sodium butyrate in the production of analytical quantities of protein from the CHO expression system, and suggests a role for sodium butyrate in commercial scale processes as well.

Pseudorabies virus as a live virus vector for expression of foreign genes.

The cDNA coding for human tissue plasminogen activator (tPA) was cloned downstream from the promoter for pseudorabies virus (PRV) glycoprotein and flanked by downstream PRV DNA. After co-transfection with PRV DNA, this plasmid recombined to insert the tPA cDNA into the viral genome. In cells infected by this recombinant virus, tPA was detected by immunoprecipitation analysis and by enzymatic activity. Since it has a wide host range but does not infect humans, PRV is a possible vaccine vector for genes from animal pathogens.

The use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukaemia virus.

The env and gag genes from feline leukaemia virus were expressed in a thymidine kinase-negative feline herpes-virus and a baculovirus. Cats were vaccinated with various combinations of these recombinant viruses and 100% protection against feline leukaemia virus challenge was achieved using an immunization schedule which utilized both env and gag products delivered at both a mucosal and systemic site.

Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system

In order to test components of feline leukaemia virus (FeLV) as subunit vaccines, we have constructed recombinant baculoviruses that express the FeLV envelope glycoprotein gp85 [Autographa californica nuclear polyhedrosis virus (AcNPV)-gp85] and the structural protein, gag (AcNPVgag). The gag protein is expressed and shed into the medium of infected cells as particles which have a buoyant density on sucrose gradients and appearance by electron microscopy similar to those of authentic FeLV virions. The gag precursor protein within the particles is not fully processed and appears to be a result of partial cleavage of the gag polypeptide. Insect cells that are coinfected with AcNPVgag and AcNPVgp85 shed particles that contain both the gag protein and the gp85 glycoprotein.

A small open reading frame in pseudorabies virus and implications for evolutionary relationships between herpesviruses

An open reading frame coding for an 11-kDa protein was located downstream from the gI gene of pseudorabies virus (PRV). This open reading frame is homologous to an open reading frame (US9) in an analogous position in herpes simplex virus and to an open reading frame (US1) in a different position in varicella zoster virus. The open reading frame encoding the 11-kDa protein is in a region known to be deleted in live attenuated vaccine strains of PRV.

Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.

The processing of pseudorabies virus glycoprotein gX in infected cells and in an uninfected cell line

Pseudorabies virus (PRV) produces a glycoprotein, gX, that accumulates in the medium of infected cells. The gX gene was expressed in Chinese hamster ovary cells (CHOgX cells) using the cytomegalovirus Towne major immediate early promoter. Like PRV-infected cells, CHOgX cells produced gX and exported it into the medium. Tunicamycin reduced the molecular weight of the gX in the medium to 89 kDa, compared with 99 kDa for gX made in the absence of drug. In the presence of tunicamycin gX produced by both PRV-infected cells and CHOgX cells was still glycosylated, as indicated by incorporation of [14C]glucosamine. The most likely form of this glycosylation is O-linked. In a pulse-chase experiment, gX first appeared in a 90-kDa form, then a 115-kDa form. This 115-kDa form is probably cleaved to give the 99-kDa form of gX that is released into the medium. The 115-kDa form was much more persistent in the PRV-infected Vero cells than in the CHOgX cells. In both cell types, gX was labeled by [35S]sulfate in the presence and absence of tunicamycin.

Immune response in pigs to Aujeszky's disease viruses defective in glycoprotein g1 or gX.

Two Aujeszkys disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszkys virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszkys disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.