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H L Heyneker

Publications and source records attributed to H L Heyneker.

At least 19 recordsLinked to original sources
PubMed1995

Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.

Here, we describe assembly PCR as a method for the synthesis of long DNA sequences from large numbers of oligodeoxyribonucleotides (oligos). The method, which is derived from DNA shuffling [Stemmer, Nature 370 (1994a) 389-391], does not rely on DNA ligase but instead relies on DNA polymerase to build increasingly longer DNA fragments during the assembly process. A 1.1-kb fragment containing the TEM-1 beta-lactamase-encoding gene (bla) was assembled in a single reaction from a total of 56 oligos, each 40 nucleotides (nt) in length. The synthetic gene was PCR amplified and cloned in a vector containing the tetracycline-resistance gene (TcR) as the sole selectable marker. Without relying on ampicillin (Ap) selection, 76% of the TcR colonies were ApR, making this approach a general method for the rapid and cost-effective synthesis of any gene. We tested the range of assembly PCR by synthesizing, in a single reaction vessel containing 134 oligos, a high-molecular-mass multimeric form of a 2.7-kb plasmid containing the bla gene, the alpha-fragment of the lacZ gene and the pUC origin of replication. Digestion with a unique restriction enzyme, followed by ligation and transformation in Escherichia coli, yielded the correct plasmid. Assembly PCR is well suited for several in vitro mutagenesis strategies.

DNA-Directed DNA Polymerase↗
PubMed1987

Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis.

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.

Amino Acid Sequence↗
PubMed1985

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme.

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.

DNA, Recombinant↗
PubMed1985

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable.

We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disulfide bonds as authentic hGH.

Cell Compartmentation↗
PubMed1984

Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli.

Plasmids have been constructed that direct the synthesis in Escherichia coli of heavy chains and/or light chains of an anti-carcinoembryonic antigen (CEA) antibody. Another plasmid was constructed for expression of a truncated form of heavy chain (Fd' fragment) in E. coli. Functional CEA-binding activity was obtained by in vitro reconstitution in E. coli extracts of heavy chain or Fd' fragment mixed with extracts containing light chain.

Amino Acid Sequence↗
PubMed1984

Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa.

A 2760-base pair DNA segment of the Pseudomonas aeruginosa strain PA103 chromosome encoding the exotoxin A (ETA) structural gene has been cloned in Escherichia coli and the nucleotide sequence has been determined. Analysis of the 5'- and 3'-flanking regions indicate that ETA is translated from a monocistronic message. Comparison of the deduced NH2-terminal amino acid sequence with that determined by sequence analysis of the secreted protein indicates that ETA is made as a 638 amino acid precursor from which a highly hydrophobic leader peptide of 25 amino acids is removed during the secretion process. Data are presented that indicate a COOH-terminal location of the ADP-ribosylation activity of the molecule. Expression of the ETA coding sequence in E. coli under control of the E. coli trp promoter, but not the ETA promoter, results in the production of enzymatically and immunologically active protein. However, most of this material appears to be neither processed nor secreted. Comparison of the ETA amino acid and nucleotide sequences to those of diphtheria toxin revealed no significant homologies, indicating that these functionally similar toxins evolved from different genes.

ADP Ribose Transferases↗
PubMed1983

Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon.

The nine base pairs immediately 5' of the initiation codon for the bovine growth hormone (BGH) structural gene have been mutagenized. The mutagenesis method employs the ligation of an ambiguously synthesized oligonucleotide duplex into a previously engineered gap in an expression plasmid for BGH. The mutation method, coupled with hybridization screening, is efficient at isolating 1 and 2 base pair changes within the targeted region. The E. coli cultures harboring the mutant plasmids were assayed for relative levels of BGH expression. The most notable result is the varied effect of substitution of G into the mRNA at various positions in this region.

Animals↗
PubMed1983

Efficient bacterial expression of bovine and porcine growth hormones.

cDNAs prepared using poly(A)mRNA from pituitaries and containing the coding sequences for bovine and porcine growth hormones (bGH and pGH) were cloned in bacteria. The primary structures of the peptide hormones derived from the nucleotide sequences of the respective cDNAs show approximately 90% homology. The cloned cDNAs were modified using synthetic DNA to construct expression vectors for efficient bacterial production of the mature animal growth hormones.

Animals↗
PubMed1981

Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin.

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.

Amino Acid Sequence↗
PubMed1980

Production of biologically active N alpha-desacetylthymosin alpha 1 in Escherichia coli through expression of a chemically synthesized gene.

Thymosin alpha 1, an immune restorative polypeptide hormone, was synthesized in Escherichia coli by using recombinant DNA cloning techniques. Based on the known amino acid sequence, a gene coding for the thymosin alpha 1 polypeptide chain was designed and enzymatically assembled from chemically synthesized oligodeoxyribonucleotide fragments. The gene was ligated into plasmid pBR322 and placed under lac operon control, and N alpha-desacetylthymosin alpha 1 was expressed as part of a beta-galactosidase chimeric protein. Cyanogen bromide cleavage of this protein gave a mixture of polypeptides, among which thymosin alpha 1 activity was detected by radioimmunoassay (RIA). The E. coli product is identical with native thymosin alpha 1 isolated from calf thymus in the amino acid sequence but lacks the N-terminal acetyl group. Results of a guinea pig migration inhibition factor (MIF) assay, a terminal deoxyribonucleotidyl transferase (TdT) assay, and radioimmunoassay indicate that the N alpha-desacetylthymosin alpha 1 produced by deoxyribonucleic acid (DNA) cloning techniques has biological activity equivalent to that of the native hormone.

Amino Acid Sequence↗
PubMed1980

Human leukocyte interferon produced by E. coli is biologically active.

A human leukocyte interferon cDNA was enzymatically synthesized, inserted into the vector pBR322, and cloned in Escherichia coli. The DNA sequence codes for a 23-amino acid signal peptide followed by an interferon polypeptide of 165 amino acids. An expression plasmid was constructed which permits the synthesis in E. coli of 2.5 x 10(8) units of interferon per litre of culture. This LeIF protected squirrel monkeys from lethal encephalomyocarditis virus infection.

Amino Acid Sequence↗
PubMed1980

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.

Base Composition↗
PubMed1980

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers.

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.

Ampicillin↗
PubMed1979

Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone.

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This 'hybrid' gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.

Base Sequence↗
PubMed1979

Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313.

By employing a system of RNA polymerase binding and restriction endonuclease digestion, we demonstrate that the region in and around the Hind III site of pBR313 and pBR322 is the promoter for the tetracycline (Tc) resistance gene(s). Furthermore, it is shown that this region was transferred intact from pSC101 during the construction of the latter plasmids. The in vitro insertion of a few base pairs at the Hind III site produces a series of "down" promoter mutations in which the level of in vivo Tc resistance is reduced. Sequence analysis of the various promoter mutations revealed significant base pair rearrangements in the region between -40 and -12 of the promoter. While these base alterations do not appear to affect the firm binding of RNA polymerase, they do affect the ability of mutant promoters to initiate transcription. These observations suggest that the region from -40 to -12, previously designated as the "recognition region", is actually involved in the process of initiation of transcription.

Base Sequence↗