David B. Evans

Immunodetection of recombinant proteins based on antibodies directed against a metal binding peptide engineered for purification by immobilized metal affinity chromatography

Recently we reported (Evans, D.B., Tarpley W.G. and Sharma, S.K. (1991) Expression and characterization of chimeric rDNA proteins engineered for purification and cleavage. Protein Expr. Purif. 2, 205-213) a genetically engineered metal binding peptide (mbp) for the purification of recombinant proteins by immobilized metal affinity chromatography (IMAC). Therefore, we have been interested in developing mbp-based immunodetection methods for these engineered proteins. To this end, the following linker peptide containing the mbp (His-Asp-His-Asp-His) was designed, synthesized and conjugated to porcine thyroglobulin: Ac-Cys-Gly-Glu-Glu-His-Asp-His-Asp-His-Pro-Phe-His-Leu. Rabbits immunized with this conjugate developed antibodies that cross-react with peptides containing the mbp sequence. A number of chimeric recombinant proteins, expressed in E. coli, with and without the mbp portion (His-Asp-His-Asp-His) of the fusion peptide (His-Asp-His-Asp-His-Pro-Phe-His-Leu) were analyzed by ELISA and immunoblotting. Results from these studies show that the anti-mbp antibodies detect chimeric proteins containing the mbp, while chimeric proteins lacking this pentapeptide were negative in both immunodetection techniques. The usefulness of this approach has also been demonstrated in following IMAC purification and enzymatic cleavage of the mbp. These immunodetection techniques utilizing anti-mbp antibodies should be applicable to other proteins engineered to contain the mbp for IMAC purification.

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.

Could angiotensin I be produced from a renin substrate by the HIV-1 protease ?

The standard angiotensin I (Ang I) radioimmunoassay for renin activity determination is a useful clinical tool for the diagnosis of high renin levels in certain cases of hypertension. It depends upon the liberation of Ang I from human plasma angiotensinogen. We considered whether a commercially available synthetic tetradecapeptide (TDP), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, would produce authentic Ang I upon incubation with protease from human immunodeficiency virus type 1 (HIV-1). This peptide is also known to be cleaved by renin at the Leu-Leu bond to yield the decapeptide Ang I. When the TDP is incubated with the HIV-1 protease, the peptide is readily hydrolyzed. Product formation is linear with respect to time and enzyme concentration. HPLC analysis of reaction products showed two new peaks, as one would expect from the cleavage of a TDP into a decapeptide and a tetrapeptide. Amino acid analysis of HPLC-purified peaks confirmed that the HIV-1 protease cleaves TDP at the Leu10-Leu11 site to produce the desired decapeptide, Ang I. Production of Ang I by the HIV-1 protease, like human renin, is inhibited in the presence of a protease inhibitor. Implications of the discovery of an HIV-1 protease substrate that produces authentic Ang I are discussed in light of a screening assay for soluble HIV-1 protease inhibitors.

Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins

BACKGROUND The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity. METHODS Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using Escherichiacoli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis. RESULTS N-terminal glutathione-S-transferase (GST) or C-terminal His(8) tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His(8) was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system. CONCLUSIONS Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems. GENERAL SIGNIFICANCE These results indicate that an Escherichiacoli-derived full-length His(8)-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase: Asymmetry in p66 subunits of the p66/p66 homodimer

A recombinant p66 form of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69‐81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV‐1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV‐1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4°C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA‐dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV‐1 RT upon incubation with the HIV‐1 protease. When the IMAC‐purified, imidazole‐free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4°C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66‐containing species were stable and displayed identical RNA‐dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole‐mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). BHAP‐resistant HIV‐1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline‐to‐leucine substitution at amino acid 236 (P236L) of HIV‐1 RT. To understand the basis for the in vitro sensitization‐resistance phenomenon, single base pair mutations at amino acid P236 in HIV‐1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV‐1 RT mutants. Active HIV‐1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV‐2 RT, were also cloned, expressed, and purified. Three BHAPs (U‐88204E, U‐87201E, and U‐90125S) and the pyridinone L‐697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L‐697,661 and resistance to the BHAPs. The HIV‐1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV‐2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV‐1 RT mutants demonstrated a direct correlation between sensitization to L‐697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L‐697,661.

Immobilized metal affinity chromatography of bacterially expressed proteins engineered to contain an alternating-histidine domain

Abstract A genetic approach to purifying chimeric recombinant proteins by immobilized metal affinity chromatography (IMAC) is described. Plasmid expression vectors have ben constructed to direct the synthesis in Escherichia coli of human immunodeficiency virus (HIV-1) reverse transcriptase (RT) designed to possess alternating histidine residues at either the N- or the C-terminus. As shown below, HIV-1 RT containing six adjacent histidines (S. K. Sharma et al. , 1991, Biotechnol. Appl. Biochem. 14 , 69–81) was also included for comparison purposes: (a) Met-Glu-(His-Glu) 4 -lle-Pro- (−2)-HIV-1 RT; (b) HIV-1 RT-Asn-Gly-(His-Asp) 4 -His-Leu; and (c) Pro-lle-(His) 6 -Pro-Phe-His-Leu-Val-Lle-His-HIV-1 RT. Protocols in which the histidine-containing domain allows the single-step purification of the chimeric proteins, under nondenaturing conditions, by IMAC on Ni 2+ immobilized to iminodiacetic acid-Sepharose are described. Most of the contaminating E. coli proteins were eliminated by washing the IMAC column with 30–35 m m imidazole. Linear gradients of imidazole utilized to recover purified chimerics resulted in elution of the alternating histidine-containing recombinant proteins between 50 and 80 m m imidazole. In contrast, the chimeric with six adjacent histidines remained bound to immobilized Ni 2+ even in the presence of 100 m m imidazole. An imidazole concentration of 300 m m was required to elute this chimeric protein from the IMAC column. It is concluded that elution by a competitive counterligand during IMAC of engineered proteins is determined by the sequence of the IMAC-specific tag. Characterization of the IMAC-purified chimerics shows that these relatively small N- or C-terminal fusions have an insignificant effect on the RNA-dependent DNA polymerase activity of HIV-1 RT. A strategy for utilizing the other alternating amino acid for stepwise trimming of the His-X dipeptides from chimeric proteins by dipeptidylpeptidases is discussed.

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s

The replacement of either Tyr‐181 or Tyr‐188 of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) by the corresponding HIV‐2 RT amino acids Ile‐181 or Leu‐188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L‐697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U‐90152S, a highly specific inhibitor (IC50, 0.29 ± 0.01 μM) of HIV‐1 RT, inhibited the recombinant Y181I and Y188L HIV‐1 RT mutants with IC50 values of 3.6 ± 0.15 μM and 0.71 ± 0.02 μM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV‐1 RT showed > 150‐fold resistance to U‐90152S. An HIV‐2 mutant containing amino acids 176–190 from HIV‐1 RT acquired full sensitivity to U‐90152S (IC50, 0.29 ± 0.01 μM). It is concluded tha simultaneous mutations at Tyr‐181 and Tyr‐188 of HIV‐1 RT promotes resistance to U‐90152S.

An ultrasensitive human immunodeficiency virus type 1 protease radioimmuno rate assay with a potential for monitoring blood levels of protease inhibitors in acquired immunodeficiency disease syndrome patients

The angiotensin I-based peptide Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Glu-Glu-Ser yields angiotensin I (Ang I) and Leu-Glu-Glu-Ser upon hydrolysis by the human immunodeficiency virus type 1 (HIV-1) protease, but not by human renin. N-terminal sequencing of the reaction products showed that the HIV-1 protease cleaved exclusively at the Leu-Leu bond. The rate of Ang I formation can be measured by a radioimmunoassay, since the parent peptide has minimal cross reactivity in this assay. The rate of enzymatic hydrolysis is maximal at pH 4.5-5.0 and at an ionic strength of 1 M. At 37 degrees C, 0.1 M Na acetate buffer, pH 5.0, 1 M NaCl, 10% glycerol, 5% ethylene glycol, 1 mg/ml bovine serum albumin, and 3 mM EDTA, the reaction obeys Michaelis-Menten type kinetics with Km = 17.2 +/- 3.5 microM and kcat = 2.30 +/- 0.33 min-1. The activity assay readily quantitates as little as 0.25 nM of HIV-1 protease. The production of Ang I by the HIV-1 protease is inhibited in the presence of a HIV-1 protease inhibitor. The newly discovered substrate is relatively insensitive to human or monkey serum. Therefore, the effect of sera from 20 patients with advanced acquired immunodeficiency disease syndrome (AIDS) on Ang I production in the above assay system was examined. Results of this study indicate that it may be possible to adapt the above Ang I-based system to determine blood levels of HIV-1 protease inhibitors in AIDS patients during clinical trials.