Dietrich Stüber

System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis

Publisher Summary This chapter presents an application of the system for high-level production in Escherichia coli and rapid purification of recombinant proteins. The advent of gene cloning, the engineering of vectors for efficient expression, and the application of fast and high-flux methods for protein purification made available many recombinant proteins of biological interest. This represented a breakthrough for the structure–function analysis of bioactive proteins and cell receptors and facilitated X-ray crystallographic studies for definition of the three-dimensional structures of these proteins. The E. coli expression system allows the high-level production of recombinant proteins in authentic form, as fusion proteins with the [His] 6 affinity tail and with mouse DHFR and the [His] 6 tail. Because of the presence of the affinity tail, proteins that are produced in a soluble form or can be solubilized with GuHCl or urea can be purified almost to homogeneity in one step by nickel chelate affinity chromatography. The purified recombinant proteins simplify the production of monoclonal and polyclonal antibodies directed against defined regions of the native proteins.

Rat Brain Glyceraldehyde‐3‐Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β‐Amyloid Precursor Protein

Abstract: Abundant senile plaques are a histological hallmark in the brain of Alzheimers disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimers disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C‐terminus of APP were recombinantly produced as fusion proteins termed “Amy” and “AmyC,” respectively. Using Amy and AmyC affinity chromatography, a 35‐kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C‐terminus of APP. This 35‐kDa protein was identified as the glycolytic enzyme gIyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.

The monofunctional glycosyltransferase of Escherichia coli is a member of a new class of peptidoglycan-synthesising enzymes

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high‐molecular‐weight penicillin‐binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacterial species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coli cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high‐molecular‐weight PBPs, this activity is not inhibited by Flavomycin.

Rhoptry-Associated Protein 1-Binding Monoclonal Antibody Raised against a Heterologous Peptide Sequence Inhibits Plasmodium falciparum Growth In Vitro

ABSTRACT Monoclonal antibodies (MAbs) specific for Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-123–711) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222 as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-123–711 MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.

The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines

An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr. As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin. Antibodies specific for this C-terminal region have been generated by 2 different methods. A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques. Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments. The antisera recognized human recombinant CR-22k overexpressed in E. coli, but not fulllength calretinin and stained fixed WiDr cells. The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin. Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins.