Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.

The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors.

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase.

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.

Identification of the active site of phosphoribosyl-dephospho-coenzyme A transferase and relationship of the enzyme to an ancient class of nucleotidyltransferases.

Malonate decarboxylase from Klebsiella pneumoniae contains an acyl carrier protein (MdcC) to which a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group is attached via phosphodiester linkage to serine 25. We have shown in the preceding paper in this issue that the formation of this phosphodiester bond is catalyzed by a phosphoribosyl-dephospho-coenzyme A transferase MdcG with the substrate 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA that is synthesized from ATP and dephospho-coenzyme A by the triphosphoribosyl transferase MdcB. The reaction catalyzed by MdcG is related to nucleotidyltransfer reactions, and the enzyme indeed catalyzes unphysiological nucleotidyltransfer, e.g., adenylyltransfer from ATP to apo acyl carrier protein (ACP). These unspecific side reactions are favored at high Mg(2+) concentrations. A sequence motif including D134 and D136 of MdcG is a signature of all nucleotidyltransferases. It is known from the well-characterized mammalian DNA polymerase beta that this motif is at the active site of the enzyme. Site-directed mutagenesis of D134 and/or D136 of MdcG to alanine abolished the transfer of the prosthetic group to apo ACP, but the binding of triphosphoribosyl-dephospho-CoA to MdcG was not affected. Evidence is presented that similar to MdcG, MadK encoded by the malonate decarboxylase operon of Malonomonas rubra and CitX from the operon encoding citrate lyase in Escherichia coli are phosphoribosyl-dephospho-CoA transferases catalyzing the attachment of the phosphoribosyl-dephospho-CoA prosthetic group to their specific apo ACPs.

Formation of catalytically active acetyl-S-malonate decarboxylase requires malonyl-coenzyme A:acyl carrier protein transacylase as auxiliary enzyme [corrected].

Functional malonate decarboxylase of Klebsiella pneumoniae is an acetyl-S-enzyme with an acetylated phosphoribosyl dephospho-CoA prosthetic group. The mdcH gene product acts as a malonyl-CoA:ACP transacylase and initiates the activation of (deacetyl)malonate decarboxylase by malonyl-transfer to the prosthetic group. The malonyl residue is subsequently decarboxylated to an acetyl residue by the decarboxylase itself. Purified malonate decarboxylase consists of the four subunits MdcA, D, E and C in an apparent 1 : 1 : 1 : 1 stoichiometry. In addition, the preparation contains substoichiometric amounts of MdcH comigrating on SDS/PAGE with MdcD. Malonate decarboxylase isolated from strains with a deletion of the mdcH gene was not activated with malonyl-CoA. Activity could be gained, however, in the additional presence of MdcH that has been synthesized in Escherichia coli and purified from inclusion bodies. Substrates for MdcH are malonyl-CoA or methylmalonyl-CoA but not acetyl-CoA. The enzyme has Km values of 16 microm for both substrates and Vmax for malonyl-CoA of 190 U.mg-1 and for methylmalonyl-CoA of 37 U.mg-1. Transfer of the methylmalonyl-residue to the prosthetic group proceeds via the covalent methylmalonyl-MdcH intermediate. The transacylase is specifically inhibited by N-ethylmaleimide, and preincubation with malonyl-CoA or methylmalonyl-CoA protects the enzyme from this inhibition.

Sequence of a gene cluster from Klebsiella pneumoniae encoding malonate decarboxylase and expression of the enzyme in Escherichia coli.

Malonate decarboxylase of Klebsiella pneumoniae consists of four different subunits and catalyzes the conversion of malonate plus H+ to acetate and CO2. The catalysis proceeds via acetyl and malonyl thioester residues with the phosphribosyl-dephospho-CoA prosthetic group of the acyl carrier protein (ACP) subunit. From a cosmid library of K. pneumoniae, a gene cluster of 9 kb has been isolated and sequenced that included the structural genes for the malonate decarboxylase. The cluster consisted of the eight consecutive genes mdcABCDEFGH and the divergently oriented mdcR gene. The intergenic regions were short (usually < 17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were found 4-10 bp before each gene. According to N-terminal protein sequencing, the mdcA, C, D and E genes encoded subunits alpha, delta, beta and gamma of malonate decarboxylase. Data bank searches for related proteins with known function revealed that MdcA represents the ACP-transferase and that MdcD and E together probably function as malonyl-S-ACP decarboxylase. MdcC is the (apo) ACP subunit. MdcB and MdcG could be involved in the synthesis and attachment of the prosthetic group. MdcH is similar to various malonyl-CoA:ACP-SH transacylases and therefore probably involved in the initial activation of the enzyme by malonylation. MdcF is a membrane protein that could function as a malonate carrier. The mdcR gene encodes a protein of the LysR regulator family. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmids harbouring the entire gene cluster including mdcR. As partial deletion of the mdcR gene impaired growth of the transformants on malonate, MdcR is probably a transcriptional regulator of the mdc genes.

H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region.

Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral envelope protein hemagglutinin (HA). In a first, kinetically distinct step, the HA polypeptide chain 2 (HA2) is inserted into the target membrane bilayer. Using hydrophobic photolabeling with the phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4(trifluoromethyl-3H-diazirin -3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, we identified the segment within HA2 that interacts with the membrane. The sole part of the HA2 ectodomain that was labeled with the membrane-restricted reagent is the NH2-terminal fusion peptide (residues 1-22). No labeling occurred within the long coiled coil region generated during the acid-induced conformational transition (Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature 371, 37-43). These data strongly suggest that the coiled coil region of HA2 does not insert into the lipid bilayer. This conclusion is at variance with the recent suggestion (Yu, Y. G., King, D. S., and Shin, Y.-K.(1994) Science 266, 274-276) that the coiled coil of HA may splay apart and insert into the target membrane, providing a mechanism by which the viral and the target membrane may come in close apposition.