Investigation into the misincorporation of norleucine into a recombinant protein vaccine candidate.

A high level of norleucine misincorporation was detected in a recombinant methionine-rich protein vaccine candidate expressed in E. coli K12. An investigation was conducted to evaluate a simple remediation strategy to reduce norleucine misincorporation and to determine if the phenomenon was either (a) due to the depletion of methionine during fermentation, (b) a result of the cultivation environment, or (c) a strain-specific effect. While supplementation with exogenous methionine improved product quality, the undesirable biosynthesis of non-standard amino acids such as norleucine and norvaline persisted. In contrast, non-standard amino acid biosynthesis was quickly minimized upon selection of an appropriate fed-batch process control strategy, fermentation medium, and nutrient feed. By expressing the same protein in E. coli BL21(DE3), it was determined that the biosynthesis of norleucine and norvaline, and the misincorporation of norleucine into the protein were primarily attributed to the use of E. coli K12 as the host for protein expression.

Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation.

BACKGROUND: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. RESULTS: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. CONCLUSIONS: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.

[Overproduction of noncanonical amino acids by Escherichia coli cells].

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.

Finding of an isoleucine derivative of a recombinant protein for pharmaceutical use.

Protein modification generally occurs by addition to the amino acid side-chains of protein at the post-translational stage, for example, by enzymatic or chemical reactions after polypeptide synthesis. Recently, the recombinant hirudin analog CX-397, a potent thrombin inhibitor, was found to contain methylated Ile residues when it was overproduced by Escherichia coli in the absence of amino acids in the culture medium. The Ile derivatives, deduced to be beta-methylnorleucine [betaMeNle; (2S, 3S)-2-amino-3-methylhexanoic acid] by systematic chromatographic analysis, do not appear to be normal post-translational modifications of the protein because Ile has no functional group in its side-chain. We, therefore, propose that betaMeNle is biosynthesized by E. coli, activated by E. coli isoleucyl-tRNA synthetase (IleRS), then incorporated into the overproduced recombinant hirudin analog. The biosynthesis of betaMeNle in E. coli is thought to occur as follows: alpha-ketovalerate is synthesized from alpha-ketobutyrate by three Leu biosynthetic enzymes, alpha-isopropylmalate synthase (IPMS) (EC 4.1.3.12), alpha-isopropylmalate isomerase (ISOM) (EC 4.2.1.33) and beta-isopropylmalate dehydrogenase (IPMD) (EC 1.1.1.85), which have broad substrate specificities. alpha-Ketovalerate is then converted to alpha-keto-beta-methylcaproate by three Ile and Val biosynthetic enzymes, acetohydroxy acid synthase (AS) (EC 4.1.3.18), acetohydroxy acid isomeroreductase (IR) (EC 1.1.1.86) and dihydroxy acid dehydratase (DH) (EC 4.2.1.9). Finally, this is converted to betaMeNle by branched-chain amino acid transaminase (EC 2.6.1.42), one of the Ile and Val biosynthetic enzymes.

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2).

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.

Evidence against the formation of 2-amino-6-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-hexanoic acid ('pyrraline') as an early-stage product or advanced glycation end product in non-enzymic protein glycation.

1. It has been suggested that 2-amino-6-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-hexanoic acid ('pyrraline') is formed as an advanced glycation end product in the Maillard reaction under physiological conditions. Antibodies were raised to caproyl-pyrraline linked to keyhole-limpet haemocyanin and were used to develop an e.l.i.s.a. and Western blotting system for the specific detection of pyrraline in samples in vivo and in vitro. 2. Human serum albumin was isolated from the serum samples of diabetic and non-diabetic subjects. Pyrraline was not detected (< 1.2 pmol) in any of the samples, indicating that it was not a major advanced glycation end product in vivo. 3. BSA was incubated separately with D-glucose and a model fructosamine, N epsilon-(1-deoxy-D-fructos-1-yl)-hippuryl-lysine, under physiological conditions for 30 days. Aliquots removed from the incubations at 5 day intervals contained no detectable pyrraline, indicating that pyrraline was not an early-stage product of the Maillard reaction in vitro. 4. The model fructosamine, N epsilon-(1-deoxy-D-fructos-1-yl)-hippuryl-lysine, was incubated at pH 7.4 and 37 degrees C for 25 days during which it degraded to hippuryl-lysine and N epsilon-carboxymethyl-hippuryl-lysine. Aliquots were removed at 5 day intervals and assayed for pyrraline. None was detected (< 23 pmol/ml) in the course of the degradation of the fructosamine (400 nmol/ml degraded), indicating that pyrraline was not a major product of the degradation of fructosamine under physiological conditions in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis and incorporation into protein of norleucine by Escherichia coli.

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.

Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli.

Interleukin-2 produced from a recombinant E. coli was found to contain as much as 19% norleucine in place of methionine in a minimal medium fermentation. Medium supplementation experiments and use of a leucine-requiring mutant host strain indicated the origin of norleucine to be de novo biosynthesis by reactions involving the enzymes of the leucine biosynthetic pathway. The misincorporation was highly suppressed by addition of either L-leucine or L-methionine to the fermentation and completely suppressed by adding both amino acids.

Norleucine accumulation by a norleucine-resistant mutant of Serratia marcescens.

A norleucine-resistant mutant was derived from an isoleucine-valine auxotroph of a leucine accumulator of Serratia marcescens. The norleucine-resistant mutant could accumulate norleucine from norvaline in the medium without the addition of methionine, which antagonized norleucine. This mutant constitutively formed homoserine-O-transsuccinylase.

Biosynthesis of norvaline, norleucine, and homoisoleucine in Serratia marcescens.

The biosynthetic pathways of norvaline homoisoleucine were examined using regulatory mutants of leucine biosynthesis in Serratia marcescens. alpha-Isopropylmalate synthetase [EC 4.1.3.12], the first enzyme of leucine biosynthesis, catalyzed the condensations of acetyl-CoA with pyruvate, alpha-ketobutyrate, alpha-ketovalerate, or alpha-keto-beta-methylvalerate as well as alpha-ketoisovalerate. These condensations were inhibited by leucine in the alpha-aminobutyrate-resistant mutant, a mutant with derepressed leucine biosynthetic enzymes. However, these condensations were coordinately desensitized in the isoleucine leaky revertant, a leucine accumulator. The formation of norvaline or homoisoleucine was greater in the leucine accumulator, but its leucine auxotroph did not form these unnatural amino acids. Thus, norvaline and homoisoleucine are considered to be formed from alpha-ketobutyrate and alpha-keto-beta-methylvalerate by the leucine biosynthetic enzymes. This view was confirmed by the findings that a norvaline accumulator could be obtained by derivation of the leucine accumulator into an isoleucine-valine auxotroph. Norleucine was also found to be formed from alpha-ketovalerate, an alpha-ketoacid corresponding to norvaline.