Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells.

The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac).

Determination of nucleotides and sugar nucleotides involved in protein glycosylation by high-performance anion-exchange chromatography: sugar nucleotide contents in cultured insect cells and mammalian cells.

We have developed a simple and highly sensitive HPLC method for determination of cellular levels of sugar nucleotides and related nucleotides in cultured cells. Separation of 9 sugar nucleotides (CMP-Neu5Ac, CMP-Neu5Gc, CMP-KDN, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Fuc, GDP-Man) and 12 nucleotides (AMP, ADP, ATP, CMP, CDP, CTP, GMP, GDP, GTP, UMP, UDP, and UTP) was examined by reversed-phase HPLC and high-performance anion-exchange chromatography (HPAEC). Although the reversed-phase HPLC, using an ion-pairing reagent, gave a good separation of the 12 nucleotides, it did not separate sufficiently the sugar nucleotides for quantification.

Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults.

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild-type Bcl-2 was compared to a Bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells.

Investigating the secretory pathway of the baculovirus-insect cell system using a secretory green fluorescent protein.

The secretory pathway is important in actively transporting proteins into the extracellular environment of eucaryotic cells. In this study a green fluorescent protein (GFP) mutant engineered to contain a secretion signal was used as a model protein in order to visualize the secretion process inside insect cells. Fluorescent microscopy indicated that significant amounts of secreted green fluorescent protein (sGFP) accumulated in High-Five, Trichoplusia ni, cells following infection with a baculovirus vector containing the gene under the polyhedrin promoter.

N-glycan patterns of human transferrin produced in Trichoplusia ni insect cells: effects of mammalian galactosyltransferase.

The N-glycans of human serum transferrin produced in Trichopulsia ni cells were analyzed to examine N-linked oligosaccharide processing in insect cells. Metabolic radiolabeling of the intra- and extracellular protein fractions revealed the presence of multiple transferrin glycoforms with molecular weights lower than that observed for native human transferrin. Consequently, the N-glycan structures of transferrin in the culture medium were determined using three-dimensional high performance liquid chromatography.

Cloning and expression of the human N-acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero- D-galacto-nononic acid biosynthetic ability.

Sialic acids participate in many important biological recognition events, yet eukaryotic sialic acid biosynthetic genes are not well characterized. In this study, we have identified a novel human gene based on homology to the Escherichia coli sialic acid synthase gene (neuB). The human gene is ubiquitously expressed and encodes a 40-kDa enzyme.

Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults.

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults.

Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors.

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12).

Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection.

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy.

Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter.

The serotonin transporter (SERT) is an N-glycosylated integral membrane protein that is predicted to contain 12 transmembrane regions. SERT is the major binding site in the brain for antidepressant drugs, and it also binds amphetamines and cocaine. The ability of various molecular chaperones to interact with a tagged version of SERT (Myc-SERT) was investigated using the baculovirus expression system.

Modifying secretion and post-translational processing in insect cells.

The baculovirus-insect cell system is a valuable tool for the expression of heterologous proteins. Due to limitations in the intracellular processing environment, however, heterologous secreted and membrane proteins are often insoluble, poorly processed, or contain 'non-human' modifications. Recent attempts to modify the insect cell secretory pathway by overexpressing processing factors have demonstrated the potential to overcome these limitations.

Overexpression of a cytosolic chaperone to improve solubility and secretion of a recombinant IgG protein in insect cells.

The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells.

II. Electrostatic effect in the aggregation of heat-denatured RNase A and implications for protein additive design.

In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the protein did not aggregate even when exposed to 75 degrees C for 24 h.

I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering.

The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65 degrees C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements.

A bacterial signal peptidase enhances processing of a recombinant single chain antibody fragment in insect cells.

The production of an antibody single chain fragment (scFv) in insect cells was accompanied by the formation of an insoluble intracellular precursor even with the inclusion of the bee melittin signal peptide. The presence of the precursor polypeptide suggests a limitation in the processing of the signal peptide so a baculovirus containing a signal peptidase from Bacillus subtilis (SipS) was constructed for expression studies. When the wild type SipS was coexpressed with scFv, preprocessed scFv fragments were no longer detected in insect cell lysates.

Thioredoxin domain non-equivalence and anti-chaperone activity of protein disulfide isomerase mutants in vivo.

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S.

Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells.

Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein (BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG.

Coexpression of molecular chaperone BiP improves immunoglobulin solubility and IgG secretion from Trichoplusia ni insect cells.

Infection of Trichoplusia ni (BTI-TN5B1-4) insect cells with a baculovirus coding for immunoglobulin G resulted in significant intracellular insolubility of the immunoglobulin chains. In order to increase the immunoglobulin solubility, the chaperone BiP was coexpressed in the insect cells using a separate baculovirus vector. This heterologous BiP was observed to associate with immunoglobulin chains in vivo and enhance the level of soluble intracellular and secreted IgG obtained from T.

Rescue of immunoglobulins from insolubility is facilitated by PDI in the baculovirus expression system.

A substantial fraction of immunoglobulin heavy and light chain polypeptides were insoluble when expressed in the baculovirus-insect cell expression system. In the presence of coexpressed heterologous protein disulfide isomerase (PDI), however, the solubility of the immunoglobulins was enhanced and IgG was secreted at higher levels from baculovirus-infected Trichoplusia ni insect cells. Pulse-chase experiments indicated that some immunoglobulin polypeptides were initially insoluble in the presence of PDI but subsequently were rescued in a soluble form competent for IgG assembly and secretion.

Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors.

Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2.

Chaperone and foldase coexpression in the baculovirus-insect cell expression system.

The BEVS has become widely utilized for production of recombinant proteins. However, protein aggregation and inefficient processing often limit yields, especially for secreted and membrane proteins. Since many proteins of pharmaceutical interest require similar posttranslational processing steps, engineering the folding, assembly, and secretion pathway may enhance the production of a wide variety of valuable complex proteins.

The kinetics of RCC1 inclusion body formation in Escherichia Coli.

The Regulator of Chromosome Condensation protein (RCC1) is located in both the soluble and inclusion body (IB) fractions of the whole cell lysate when expressed in Escherichia coli BL21 (pLysS) at temperatures below 30 degrees C. When bacterial growth was carried out at 20 degrees C, the majority of the RCC1 remained soluble up to 5.5 h postinduction, When the temperature was raised to 25 degrees C, RCC1 IB was dominant by 1.

Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hantaan virus.

The formation of Hantaan (HTN) virus nucleocapsid-like structures (NLS) or virus-like particles (VLP) from expressed gene products was investigated in two eukaryotic systems. Baculovirus expression of the HTN virus small segment (S), which encodes the viral nucleocapsid protein, resulted in assembly of NLS inside infected insect cells. The NLS and authentic ribonucleocapsids, prepared by detergent disruption of HTN virions, had similar sedimentation characteristics and morphologies, and were recognized by HTN virus N-specific antibodies.