Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties.

A combinatorial mutagenesis strategy was used to create a collection of nearly 500 variants of human interleukin 3 (IL-3), each with four to nine amino acid substitutions clustered within four linear, nonoverlapping regions of the polypeptide. The variants were secreted into the periplasm of Escherichia coli and supernatants were assayed for IL-3 receptor-dependent cell proliferation activity. Sixteen percent of the variants, containing "region-restricted" substitutions, retained substantial proliferative activity through two rounds of screening. A subset of these was combined to yield variants with substitutions distributed through approximately half of the polypeptide. With one exception, "half-substituted" variants exhibited proliferative activity within 3.5-fold of native IL-3. A subset of the "half-substituted" variants was combined to yield "fully substituted" IL-3 variants having 27 or more substitutions. The combination of the substitutions resulted in a set of polypeptides, some of which exhibit increased proliferative activity relative to native IL-3. The elevated hematopoietic potency was confirmed in a methylcellulose colony-forming unit assay using freshly isolated human bone marrow cells. A subset of the multiply substituted proteins exhibited only a modest increase in inflammatory mediator (leukotriene C4) release. The molecules also exhibited 40- to 100-fold greater affinity for the alpha subunit of the IL-3 receptor and demonstrated a 10-fold faster association rate with the alpha-receptor subunit. The multiply substituted IL-3 variants described in this study provide a unique collection of molecules from which candidates for clinical evaluation may be defined and selected.

Optimization of heterologous protein production in Escherichia coli.

Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins. Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure. The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.

Analysis of novel streptavidin-binding peptides, identified using a phage display library, shows that amino acids external to a perfectly conserved consensus sequence and to the presented peptides contribute to binding.

Streptavidin-binding peptides containing the consensus amino acid sequence motif EPDW were identified using a phage display library. Phage presenting peptides containing these sequences bound streptavidin in a biotin-sensitive fashion and could be eluted with biotin. The previously identified 'streptag' peptide sequence (AWRHPQGG) competed with phage presenting the EPDW consensus sequence for streptavidin binding. Furthermore, the EPDW sequence has two amino acids in common with yet another previously identified streptavidin-binding sequence, GDWVFI, which has similar biochemical properties. Binding inhibition studies revealed that residues flanking EPDW, as well as residues of the modified phage pIII product to which displayed peptides are fused, contributed to streptavidin binding. The derivation of small molecules based on the structure of peptides selected using display methods is a potentially important application of phage display technology. The relevance of the observations made here for that application are discussed. Finally, a group of 'nuisance' peptides of the consensus sequence WHWWXW, whose binding specificity has not been fully elucidated, but which have been isolated in a number of biopanning experiments, including those that do not utilize streptavidin, are also described.

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody.

Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteine-constrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.

Saturation mutagenesis of human interleukin-3.

A deletion variant of human interleukin-3, hIL-3(15-125), was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3(15-125). Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3(15-125) could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by the receptor.

A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli.

Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for 'bacmid' recombinants at 44 degrees C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding beta-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of > or = 10(5) per micrograms input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.

High level expression and refolding of mouse interleukin 4 synthesized in Escherichia coli.

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.

Recent advances in heterologous gene expression in Escherichia coli.

Recent advances in protein expression in E. coli have focused primarily on the enhancement of protein quality. Problems in mRNA translation such as inefficient initiation, mistranslation, frame-shifting and frame-hopping can often be addressed by altering heterologous gene-coding sequences. Fusion technology can also be used to address problems in translational initiation, the authenticity of amino-terminal amino acids, in vivo protein activity and protein purification. Accessory molecules, such as chaperones, are increasingly used to enhance protein quality in vivo and in vitro. E. coli has recently gained wide use as a host both for the engineering of proteins with altered activities and for the creation of multi-functional hybrids.

Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli.

The construction and purification of recombinant baculovirus vectors for the expression of foreign genes in insect cells by standard transfection and plaque assay methods can take as long as 4 to 6 weeks. This period can be reduced to several days by using a novel baculovirus shuttle vector (bacmid) that can replicate in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. The bacmid is a recombinant virus that contains a mini-F replicon, a kanamycin resistance marker, and attTn7, the target site for the bacterial transposon Tn7. Expression cassettes comprising a baculovirus promoter driving expression of a foreign gene that is flanked by the left and right ends of Tn7 can transpose to the target bacmid in E. coli when Tn7 transposition functions are provided in trans by a helper plasmid. The foreign gene is expressed when the resulting composite bacmid is introduced into insect cells.

High-level production of active HIV-1 protease in Escherichia coli.

High levels of active HIV-1 protease (PR) were produced in Escherichia coli, amounting to 8-10% of total cell protein. High production levels were achieved by altering the following parameters: (1) codon preference of the coding region, (2) A+T-richness at the 5' end of the coding region, and (3) promoter. To circumvent the toxicity of HIV-1 PR in E. coli, the gene was expressed as a fusion protein with two different proteolytic autocleavage sequences. In both the cases, the fusion protein could be cleaved in vivo to give an active molecule with the native sequence at the N terminus.

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.

Effects of signal peptide changes on the secretion of bovine somatotropin (bST) from Escherichia coli.

Bovine somatotropin (bST) was secreted from Escherichia coli at moderate levels of 1-2 micrograms/ml/OD using expression vectors in which the bST gene was fused to the lamB secretion signal. To study the secretion properties of bST in E.coli further, two approaches for modifying the secretion signal were employed. In the first case, fusion proteins were constructed with six alternative bacterial secretion signals: three from E.coli proteins (HisJ, MalE and OmpA), two from bacteriophage proteins (M13 coat protein and PA-2 Lc) and one from the chitinase A protein of Serratia marcescens. The results, as monitored by Western blot analysis of both total cell protein and the periplasmic fraction, showed that these changes in the secretion signal did not significantly affect the secretion properties of bST. In the second approach, a library of random mutations was created in the lamB secretion signal and 200 independent clones were screened. The level of secreted bST was determined by growing individual clones in duplicate in microtiter wells, inducing protein expression and measuring the bST released by osmotic shock using a particle concentration fluorescent immunoassay. The secretion properties of several novel variants in the LamB signal peptide are presented.

Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli.

The effect of overexpression of the heat shock chaperone genes dnaK and groESL on heterologous protein production in Escherichia coli was examined, using a set of related human procollagenase proteins. A diverse range of effects on protein solubility, secretion, and accumulation was observed, and these effects were highly dependent on the particular chaperone/procollagenase pairing involved. Both chaperones caused a large increase in the apparent solubility of a fusion of the LamB signal peptide to procollagenase. GroESL had no effect on the accumulation of mature (secreted) procollagenase, while DnaK suppressed secretion considerably. In the absence of a signal peptide, overexpression of either chaperone resulted in a dramatic increase in both solubility and accumulation of procollagenase. The 10-fold increase in accumulation was associated with an increase in in vivo protein half-life.

Secretion of active bovine somatotropin in Escherichia coli.

We have expressed a chimeric protein, comprising the LamB secretion signal sequence fused to mature bovine somatotropin (bST), in Escherichia coli. Plasmid constructs with the recA promoter showed significant protein accumulation prior to induction and cell lysis occurred after induction. In contrast, the lacUV5 promoter was tightly regulated. With the lacUV5 promoter, temperature and inducer concentration had significant effects on the total amount of recombinant protein produced and the fraction processed to mature bST. Quantitation of bST from shake flask cultures showed that 1-2 micrograms/ml/OD550 could be released from the periplasm by osmotic shock. N-terminal sequence analysis of the purified protein indicated that the majority of the secreted bST was correctly processed. The bST present in the osmotic shock fraction was judged to be correctly folded by comigration with oxidized methionyl-bST standard on a non-reducing polyacrylamide gel and activity in a bovine liver radioreceptor assay. These results provide a rapid method to produce bST for use in structure-function studies.

Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.

A broad host-range expression plasmid was constructed comprising the incQ replicon, the recA promoter from Escherichia coli and the g10-L ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple "consensus" RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.

Compartmentalization of mammalian proteins produced in Escherichia coli.

We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents. These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta. Efficient expression of these proteins in E. coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7. In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass. Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction. Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g. phosphoglycerate dehydrogenase). Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing. The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase. Together the data suggest that osmotic shock fractionation of E. coli may facilitate the purification of functional foreign proteins produced in this prokaryote. They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E. coli.

Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.

A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli.

We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."

Expression, purification, and in vivo activity of atrial natriuretic factor prohormone produced in Escherichia coli.

Atrial muscles of the heart are known to produce polypeptide hormones called atrial natriuretic factors (ANF) which have potent diuretic and hypotensive action. These hormones are synthesized as a larger protein precursor called pro atrial natriuretic factor or proANF which contains the biologically active ANF sequences at its C-terminus. Rat proANF (representing amino acids -1 to 128 of the coding sequence) was expressed in a soluble form in Escherichia coli. A simple purification procedure was developed which consists of boiling E. coli cell extracts in 1 M acetic acid and subjecting the supernatant to reversed-phase HPLC. The effect of intravenous administration of the purified recombinant proANF on mean arterial blood pressure was examined. The displacement dose-response curves obtained demonstrated that proANF exhibits similar, albeit less potent, physiological activity than ANF.