Michael R. Schlittler

High-yield production of a human therapeutic protein in tobacco chloroplasts

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

Formation of isoaspartate 99 in bovine and porcine somatotropins

Asparagine 99 in bovine (BST) and porcine somatotropins (PST) was converted to an isoaspartate residue during incubation at neutral or alkalinepH. Isoaspartate 99 BST or isoaspartate 99 PST was resolved from the normal somatotropin by reversed-phase high-performance liquid chromatography (HPLC). The altered peptide of residues 96–108 which contains isoaspartate 99 was detected by tryptic peptide mapping of the modified BST or PST. Amino acid sequencing, amino acid analysis, mass spectrometry, and co-elution with a chemically synthesized peptide containing isoaspartate 99 were used to demonstrate the existence of isoaspartate in the modified peptides. Peptide bond cleavage between Asn 99 and Ser 100 also occurred during incubation of BST and PST at neutral or alkalinepH. This chemically cleaved product was resolved on reversed-phase HPLC from both the isoaspartate 99 and normal somatotropin molecules.

Increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli

In Escherichia coli and most other microorganisms, peptide synthesis is started at methionine start codons which are read only by N-formyl-methionine-tRNA. The formyl group is normally removed from the N-terminal Met residue of the peptide by peptide deformylase (PDF). However, it has been observed that overproduction of proteins in recombinant bacteria often yields protein products which are incompletely deformylated. Certain proteins could be poor substrates for PDF and exhibit incomplete deformylation, particularly when they are overproduced. Strains of E. coli which overproduce bovine somatotropin (BST) have a significant fraction of the BST with the formyl group retained. The PDF gene was isolated and positioned into a BST production vector in such a way that the BST and PDF genes were coexpressed. In strains containing this coexpression vector, the levels of PDF were increased and formylated BST was undetectable.

Structural characterization of the two refold dimers of recombinant bovine somatotropin (bST).

Two major dimers are generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin (bST). These dimers represent the major part of the inactive high molecular weight species that are formed in this process. The structures of the two dimers are unambiguously determined by peptide mapping using trypsin, thrombin cleavage, and selective DTT reduction experiments. Results indicate that the formation of both dimers involves the large disulfide loop cysteines. The latter-eluting dimer from RP-HPLC, previously reported as a large loop concatenated dimer, was revised to be an antiparallel disulfide-linked dimer. On the other hand, the first eluting dimer is a concatenane in which two monomers are held together by the interlocking of the two large disulfide loops.

Isolation of Escherichia coli synthesized recombinant proteins that contain ε-N-acetyllysine

Publisher Summary This chapter describes the presence of significant amounts of ɛ-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. ESMS, amino acid sequencing and amino acid analyses demonstrate the presence of ɛ-N-acetyllysine in two recombinant proteins; bovine placental lactogen and human tissue factor pathway inhibitor. These data establish that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E.coli . Formation of ɛ-N-acetyllysine in eukaryotic systems involves a post-translational mechanism in which the acetyltransferase uses acetyl-CoA as the source of the acetyl group. It may be possible that acetylation of lysines occurs by a chemical mechanism with acetyl-CoA or some other metabolic intermediate providing the source of the acetyl group. The chapter explains that acetylation of lysines is an important modification, which can occur during the expression of recombinant proteins expressed in E.coli. However, investigations into the effects of fermentation conditions on the level of ɛ-N-acetyllysine formation may lead to a better understanding of this event.

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.

Development and optimization of a SE-HPLC method for proteins using organic mobile phases

Publisher Summary This chapter describes the development and optimization of an organic mobile phase size exclusion high-performance liquid chromatography (SE-HPLC) method using organic mobile phases for several proteins. With an acetonitrile/trifluoroacetic acid (TFA) mobile phase, non-ideal SE-HPLC (ionic, hydrogen bonding and hydrophobic interactions) is minimized. SE-HPLC is the method to monitor removal of the oligomers during purification of the proteins. Most SE-HPLC methods for proteins use aqueous mobile phases containing salts or chaotropic agents, such as sodium dodecyl sulfate (SDS) or urea. Hydrophobic and ion-exchange interactions occurring between the protein and the chromatography resin with these mobile phases may result in nonideal size exclusion chromatography. Limited sample solubility and non-covalent oligomer formation may also occur with these mobile phases resulting in inconsistent performance of the method. The chapter demonstrates that acetonitrile/TFA mobile phases can be effectively used for analyzing several different proteins.