Che-Shen C. Tomich

Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofMr ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

Enhancement of heterologous polypeptide expression by alterations in the ribosome-binding-site sequence

Abstract In our studies to improve the expression of bovine growth hormone in E. coli , we have observed that the production of the hormone can be greatly increased by three similar AT-rich ribosome-binding-site sequences. Thus the low level expression of the hormone at less than 1% of the total cellular protein can be enhanced to about 15–30% by simply enriching the sequence flanking the Shine-Dalgarno site with adenine- and thymine-containing deoxyribonucleotides. These AT-rich ribosome-binding-site sequences enhance the expression of porcine growth hormone in a similar manner. These sequences also increase the moderate expression (about 5%) of tumour necrosis factor to high levels of about 30%.

Single-Step Purification and Biological Activity of Human Nerve Growth Factor Produced from Insect Cells

Abstract: Human nerve growth factor (NGF) was cloned and engineered for expression in a baculovirus‐infected Spodoptera frugiperda (SF‐9) insect cell system. Culture supernatants contained 2–3 mg/L of recombinant human NGF. The human NGF produced by this system was purified to apparent homogeneity with a single‐step affinity chromatography procedure using a high‐affinity monoclonal antibody originally raised against murine NGF. The purification procedure yielded 1–2 mg of pure, human NGF per liter of culture supernatant; i.e., approximately 60% recovery of the human NGF originally released into the culture medium. Although the gene transacted into the SF‐9 cells coded for pro‐NGF, the NGF recovered after purification was > 95% fully processed, mature protein. The KD for the affinity of the pure, recombinant human NGF for NGF receptor in PC12 membranes is 0.20 ± 0.05 nM. Activation of neurite outgrowth in PC12 cells occurs with ED50 values of 85 ± 20 pM and 9.6 ± 1.5 pM for a 3‐day primary response and a 1‐day secondary response, respectively. The pure, recombinant human NGF also stimulates a significant increase in dopamine content of PC12 cells with an ED50 of 5.8 ± 2.7 pM. These binding and biological activation properties are consistent with values observed using murine NGF purified from sub‐maxillary glands.

High-level expression of human renin in Escherichia coli

Abstract The cDNA sequence for human renin was modified for use in the expression of the mature protein in E. coli . This was accomplished by the removal of the 5′ untranslated region and sequences coding for the signal peptide and a portion of the mature protein. An oligonucleotide linker was inserted which supplied the deleted coding information for the mature protein in a form optimized for translation in E. coli , in addition to an initiation codon. The modified gene was cloned into an expression vector consisting of the promoter from the tryptophan operon of E. coli and trp L Shine-Dalgarno sequence. In an appropriate host strain the expressed protein is the most prominent species present, and accounts for at least 10% of the total cellular protein. The expressed protein was verified to be renin by its molecular weight, ability to bind a renin antibody, and N-terminal amino acid sequence.

Secretion of active truncated CD4 into Escherichia coli periplasm

SummaryA truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.

Gene synthesis, E. coli expression and purification of the bovine growth hormone releasing factor analog, (Leu27,Hse45) bGRF

Abstract The DNA sequence coding for an analog of bovine growth hormone releasing factor (bGRF), having the methionine at position 27 substituted by leucine, was chemically synthesized with flanking methionine codons. Tandem ligations of this sequence allowed the monomer or fusion proteins of 2, 4, or 8 bGRF units to be expressed in E. coli from a pBR322-based vector. Although the monomer and dimer genes do not generate detectable products by SDS-PAGE, the fusion products of 4 and 8 bGRF accumulate as insoluble aggregates representing over 20% of the cellular protein; there appears to be decreased protease susceptibility as the fusion protein increases in size. The inclusion body material was solubilized in formic acid and octameric bGRF was treated with cyanogen bromide to release the monomer containing the natural amino-terminal tyrosine and an unnatural carboxy-terminal homoserine lactone. The reaction conditions represented a compromise between minimizing a formic acid-dependent modification of bGRF and achieving complete digestion. About 150 g of the bGRF analog were isolated at ≥99% purity using ion exchange and preparative reverse-phase chromatography. Intravenous injection of this peptide into steers stimulates somatotropin release as measured by significant increases in serum concentrations of the hormone.

Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template

A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

Synthesis of a Gene Coding for Human Vasoactive Intestinal Peptide (VIP)

Abstract A gene coding for human Vasoactive Intestinal Peptide (VIP) was designed as a double-stranded 99 base pair DNA sequence. The sixteen fragments of the gene were chemically synthesized using a solid-phase phosphoramidite triester coupling approach and enzymatically assembled using T4 DNA ligase. The resulting gene was cloned into pBR322 and sequenced using the Maxam-Gilbert sequencing procedure.

A Two Step Purification of Recombinant Human Interleukin-1β Expressed in E. Coli

Recombinant human interleukin-1β has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1β is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1β (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 × 107 units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1β sera.