Larry B. Tsai

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.

The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell densityEscherichia coli fermentations

SummaryTwo kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period.

Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E. coli

Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E. coli have been detected. Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC. The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry. In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E. coli.

Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor

Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.

Use of a modified tryptophanase promoter to direct high-level expression of foreign proteins in E. coli.

We have modified the tryptophanase promoter (PtnaA) for use as a temperature-independent promoter for the production of recombinant proteins. Although any protein will have a temperature range in which its expression is optimal, we find the tryptophanase promoter functions at all physiologically relevant temperatures (20 degrees C to 42 degrees C). Induction at temperatures below 37 degrees C avoids eliciting the heat-shock response and may favor the production of protein in the soluble state. A short segment of the E. coli tnaA promoter containing the catabolite gene activator protein (CAP) binding site but no tryptophan-responsive elements was used to direct synthesis of various proteins. Conditions for high cell density fermentation and induction control were developed. Expression was induced by depletion of glucose and was maximal when an alternative nonrepressing carbon source was supplied. Expression of certain proteins was tightly controlled; however, pre-induction expression was observed with other reporter genes. The tnaC leader portion of the tnaA promoter was found to reduce pre-induction expression in the presence of glucose, although maximal expression was observed only in the absence of this region. The effect of temperature on expression of several recombinant proteins was investigated. Although some proteins were produced only in inclusion bodies as insoluble material, the production of one protein in soluble form was clearly temperature dependent.

Protein Drug Manufacturing

Recombinant DNA technology has opened new avenues for producing many useful therapeutic proteins, such as hormones, growth factors, and interferons, in commercial quantities. To economically produce therapeutic proteins at the commercial scale, while controlling product quality, requires three general steps. First, an effective strategy for maximizing recombinant gene expression must be devised. Next, an efficient fermentation process must be developed. Finally, robust, effective protein recovery and purification processes must be developed.

E. coli-expressed human neurotrophin-3 characterization of a C-terminal extended product

Publisher Summary In the development of proteins as therapeutic agents, it is crucial to ensure the homogeneity of the potential products; however, over-expression of recombinant proteins in E.coli often leads to translational errors as well as post-translational modifications. This chapter describes the E.coli expression of human neurotrophin-3 (NT-3), a member of the nerve growth factor (NGF) family of neurotrophic factors, and discusses the characterization of this higher molecular weight product, and shows that it is due to misreading through the UGA codon by tryptophan incorporation. The chapter shows that when the cloned DNA is terminated with the codon UGA, both the mature protein and a higher molecular form are produced in the inclusion bodies. Examination of the endoproteinase Lys-C map of the higher molecular weight material demonstrates that the peptides corresponding to the C-terminus are absent and a new peptide is generated. N-terminal analysis of the peptide gives IGRTWGSADK. Therefore, the C-terminus of the protein has been extended with the peptide WGSADK. This sequence matches perfectly with the translated cloned DNA sequence, before another in-frame termination codon (UAA) is met. The chapter explains that the higher molecular weight form of NT-3 is caused by reading through of the UGA stop codon by tryptophan incorporation.

Assessing post-induction cellular response in a recombinant E. coli lactose-inducible system by monitoring β-galactosidase levels

SummaryA synthetic lactose-inducible promoter was chosen to study host cell responses to the over-expression of heterologous genes. Fermentations were conducted to compare the effect of induction strategies on the synthesis of β-galactosidase versus the production of recombinant protein. The levels of lactose, IPTG and glucose during induction were manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In addition, the involvement of the gal operon in lactose metabolism was also explored in order to optimize lactose transport and utilization during induction.