In Escherichia coli high-level production of some heterologous proteins (specifically, human prorenin, renin, and bovine insulin-like growth factor 2) resulted in the induction of two new E. coli heat shock proteins, both of which have molecular masses of 16 kDa and are tightly associated with inclusion bodies formed during heterologous protein production. We named these inclusion body-associated proteins IbpA and IbpB. The coding sequences for IbpA and IbpB were identified and isolated from the Kohara E. coli gene bank. The genes for these proteins (ibpA and ibpB) are located at 82.5 min on the chromosome. Nucleotide sequencing of the two genes revealed that they are transcribed in the same direction and are separated by 110 bp. Putative Shine-Dalgarno sequences are located upstream from the initiation codons of both genes. A putative heat shock promoter is located upstream from ibpA, and a putative transcription terminator is located downstream from ibpB. A temperature upshift experiment in which we used a wild-type E. coli strain and an isogenic rpoH mutant strain indicated that a 32-containing RNA polymerase is involved in the regulation of expression of these genes. There is 57.5% identity between the genes at the nucleotide level and 52.2% identity at the amino acid level. A search of the protein data bases showed that both of these 16-kDa proteins exhibit low levels of homology to low-molecular-weight heat shock proteins from eukaryotic species.One of the most widely studied and utilized hosts for heterologous gene expression is the gram-negative bacterium Escherichia coli. Numerous genes encoding prokaryotic, viral, or eukaryotic proteins have been cloned and expressed in E. coli. High-level accumulation of some proteins in E. coli results in the formation of intracellular electron-dense protein aggregates that are referred to as inclusion bodies (17,23,43). In some instances, these aggregates appear to be refractile when they are examined by phase-contrast microscopy (similar to gram-positive endospores); such aggregates have been termed refractile bodies (17). Some examples of cloned proteins which, when they are overexpressed, result in the formation of refractile bodies include E. coli proteins, such as aspartase (28), the sigma subunit of RNA polymerase (11), and penicillin acylase (37), and eukaryotic proteins, such as human immunodeficiency virus reverse transcriptase (21), poliovirus 3C protease (16) human insulin (8), human renin (15, 38), and bovine growth hormone (36).In this paper we describe two 16-kDa proteins that were produced during high-level expression in E. coli of some heterologous proteins, such as human renin, prorenin, and bovine insulin-like growth factor 2 (bIGF-2). The 16-kDa proteins were tightly associated with inclusion bodies which were formed in cells that overproduced these heterologous proteins. The genes encoding the 16-kDa proteins were cloned from the Kohara E. coli gene bank (18) by using degenerate oligonucleotide probes generated from the amino acid sequences of th...

Inter-alpha-trypsin inhibitor (ITI) is a 180 kd serine proteinase inhibitor found in human serum. Treatment of 180 kd ITI with trypsin releases a 30 kd fragment (HI-30) which contains the anti-proteolytic activity of the high molecular weight form. We have isolated a cDNA clone from a human liver library which codes for HI-30, and have determined its DNA sequence. The mRNA not only codes for HI-30 but also another serum protein, alpha-1-microglobulin, which has not been previously associated with ITI or HI-30. The alpha-1-microglobulin sequence is found in the amino-terminus of the protein and is preceded by a signal sequence. HI-30 is found at the carboxy-terminus. The two protein sequences are separated by two arginine residues.

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 . 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.Human interleukin-3 (hIL-3) 1 is a multilineage hematopoietic cytokine acting in the bone marrow to promote the growth of most lineages of blood cell precursors (1). Recently, exogenously administered hIL-3 has shown promise for the clinical relief of neutropenia and thrombocytopenia induced by cancer chemotherapy (2, 3). Sequence homology comparisons of hIL-3 with other proteins indicate that it is a member of the hematopoietic cytokine family (4 -6) and that it adopts a four-␣-helix bundle topology (7-10). The protein binds to a receptor comprising at least two nonidentical subunits (11, 12). Although the precise nature of interaction between hIL-3 and its receptor is not known, studies using site-specific mutants have shed some light on which portions of the protein are important for function (8,(13)(14)(15)(16)(17). In particular, mutagenesis of the adjacent helices A and D indicate that these regions are important for interaction with the receptor. This is similar to the findings for human interleukin-5 and human granulocyte-macrophage colony stimulating factor, whose receptors share a common ␤ subunit with the hIL-3 receptor (11,18,19). Other members of the hematopoietic cytokine family also have important residues in helices A and D (19 -23) and in helix C (20,22,24,25).In this paper we have undertaken an extensive mutagenesis of hIL-3 in order to discover mutants with enhanced proliferative activity and to define residues necessary for activity. Although alanine scanning mutagenesis has been successfully used to derive structure-activity information (20, 26 -32), we chose to perform a more extensive mutagenesis, permitting the incorporation of any of the possible 19 substitutions (33). MATERIALS AND METHODS Production of hIL-3 and Variants in the Escherichia coli Cytoplasm-General techniques for manipulation of DNA are described elsewhere (34). The hIL-3 gene (35) was obtained from British Biotechnolo...

Polymyxin B is an antibiotic that kills sensitive cells by disrupting their membranes. We have cloned a wild-type yeast gene that, when present on a high-copy-number plasmid, renders the cells resistant to the drug. The nucleotide sequence of this gene is presented. A single open reading frame within the sequence has the potential to encode a polypeptide (molecular mass of 77.5 kDa) that shows strong homologies to polypeptides of the protein kinase family. The gene, PBS2, located on 'chronmosome X, is not allelic to the previously described PBS) gene (where PBS signifies polymyxin B sensitivity). Although pbs) mutations confer resistance to high levels of polymyxin B, double mutants, pbs) pbs2, are not resistant to the drug, indicating that PBS2 is essential for pbs) activity.Models based on the proposed protein kinase activity of the PBS2 gene product are presented to explain the interaction between PBSI and PBS2 gene products involved in conferring polymyxin B resistance on yeast cells.

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric protein...