Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.     

Assessment with magnetic resonance imaging of response to radiotherapy for tongue and mouth floor tumors

The recombinant outer-surface protein A with an N-terminally truncated form (des-Cys1-OspA) from the Lyme disease spirochete Borrelia burgdorferi was expressed in Saccharomyces cerevisiae at high production levels. Since the recombinant vaccine candidate expressed in Escherichia coli exhibits low production yields and the purification of lipoproteins appears to be difficult, we have investigated the secretion of a soluble recombinant OspA in the yeast S. cerevisiae. In this way, a Leu+ derivative of S. cerevisiae cI3ABYS86 was used as the host strain transformed with an expression plasmid containing the gene encoding des-Cys1-OspA and driven by the MF alpha 1 promoter. The fed-batch culture results revealed that an efficient secretion of des-Cys1-OspA is obtained with a high production level of about 2.1 g l-1 at a cell density of 101 g l-1 cell dry weight. The accumulation of recombinant protein in the supernatant exceeds 6% of the total yeast proteins when estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Moreover, des-Cys1-OspA showed lower solubilities at high cell densities and, as a consequence, a fraction of the recombinant protein precipitated. An internal cleavage of the MF alpha 1 pro::des-Cys1-OspA precursor was also detected. However, in this case the cleavage occurred at a frequency such that the large amounts of the secreted des-Cys1-OspA could be employed for the evaluation of an immunogenic effect on animal immunization. These studies will extend the knowledge of the usefulness of OspA as a vaccine for Lyme borreliosis.     

Affinity-assisted in vivo folding of a secreted human peptide hormone in Escherichia coli

We show that coexpression of a specific binding protein in Escherichia coli can significantly improve the relative yields of correctly folded human insulin-like growth factor I (IGF-I). A glutathione redox buffer was used during growth to allow formation and breakage of disulfide bonds in the periplasm of the bacterial host. Both the binding protein and the peptide hormone were produced as affinity fusions, which allowed purification of the in vivo formed heterodimer by alternative affinity purification methods. The use of affinity-assisted in vivo folding has general implications for expression, folding, and purification of recombinant proteins.     

Repeated fed-batch operations for microbial detoxification of mercury using wild-type and recombinant mercury-resistant bacteria

A wild-type mercury-resistant strain Pseudomonas aeruginosa PU21 (Rip64), and an Escherichia coli PWS1 strain genetically engineered to harbor mercury resistance were examined for their capacity to detoxify soluble mercuric ions with repeated fed-batch operations. The specific mercury detoxification activity for the two strains at different initial mercury concentrations was determined by resting-cell experiments. The fed-batch operations were conducted with different initial culture volumes (Vo), inoculum sizes (Xo), and different mercury feeding rates (FHg) to investigate the effects of those operation parameters on the performance of mercury detoxification. The results showed that the wild-type and the recombinant strains had an optimal specific activity of 5 x 10(-7) and 8 x 10(-8) micrograms cell-1 h-1, respectively. In fed-batch operation for P. aeruginosa PU21, under the conditions of Vo = 400 ml and Xo = 4.5-4.8 x 10(9) cells ml-1 the overall mercury detoxification efficiency (eta) for FHg = 16.9 mg Hg h-1 was 5.26 mg Hg l-1 h-1, nearly 35% higher than that for a lower FHg (11.7 mg Hg h-1). Among the three initial culture volumes examined in this study, the highest eta (5.60 mg Hg l-1 h-1) was obtained when Vo = 1200 ml and FHg = 16.9 mg Hg h-1. It was also found that an inoculum size higher than 4.0 x 10(9) cells ml-1 enabled a stable fed-batch operation, while as the inoculum was reduced to around 1.6 x 10(9) cells ml-1, the mercury feeding caused severe cell death, leading to an unsuccessful fed-batch operation. In the fed-batch operation for E. coli PWS1 strain with Vo = 1200 ml and FHg = 16.9 mg Hg h-1, the mercury detoxification efficiency was 3.07 mg Hg l-1 h-1, only 54% of that for the wild-type P. aeruginosa PU21 strain under the same operating conditions. It was also noticed that the operation with E. coli PWS1 became less efficient at the second fed-batch cycle due to plasmid instability of the recombinant strain.     

Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli

The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C-terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C-terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection.     

Glucocorticoids increase retinoid-X receptor alpha (RXRalpha) expression and enhance thyroid hormone action in primary cultured rat hepatocytes

A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9. 0 +/- 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.     

Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.     

Recombinant allergen Lol p II: expression, purification and characterization

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.     

Overexpression in Escherichia coli and affinity purification of chick kidney ferredoxin

Vertebrate ferredoxins are 12-14-kDa iron-sulfur proteins, some of which transfer electrons to mitochondrial cytochrome P450s. The function of many of these cytochrome P450s is to catalyze stereospecific hydroxylation of endogenous steroids. As part of our interest in the kidney mitochondrial 1 alpha-hydroxylation of 25-hydroxyvitamin D3, we have constructed an expression plasmid coding for a fusion protein containing the chick kidney ferredoxin. We subcloned chick kidney ferredoxin cDNA, obtained from our vitamin D-deficient chick kidney library by polymerase chain reaction (Brandt, M. E., Gabrik, A. H., and Vickery, L. E. (1991) Gene (Amst.) 97, 113-117) into Qiagen's pQE9, which contains an N-terminal 6xHis tag (peptide sequence for 6 adjacent histidines present in the recombinant proteins). The coding sequence was preceded by a factor Xa cleavage site. The resulting plasmid, pQTcFdx, was overexpressed in Escherichia coli, and the soluble fusion protein was purified from the cell lysate in one step by Ni(II)-nitrilotriacetic acid-agarose chromatography. We obtained 7-10 mg of greater than 99% homogeneous fusion protein from a 1-liter culture and 4-6 mg of mature ferredoxin cleaved by factor Xa. The fusion protein possessed an absorption spectrum and an electron paramagnetic resonance spectrum quantitatively indistinguishable from those published for ferredoxin purified from adrenal glands and placenta or expressed in E. coli with another vector. The fusion protein was active in supporting the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 in a reconstitution assay of a solubilized, partially purified preparation of cytochrome P450 from vitamin D-deficient chick kidney. We conclude that the procedure described here is an efficient way to produce and purify vertebrate ferredoxin; the [2Fe-2S] cofactor is assembled in vivo and effectively incorporated into the fusion protein in E. coli; slight alterations at the N terminus do not alter incorporation of the [2Fe-2S] cofactor or the biological activity of ferredoxin, and post-translational modifications, such as phosphorylation, are not an absolute requirement for ferredoxin electron transporting activity. The recombinant ferredoxin can be used for physical studies and other structure-function studies.     

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.     

Thioredoxin fusion/HIV-1 protease coexpression system for production of soluble human IL6 in E. coli cytoplasm

In this paper, thioredoxin (TRX) fusion expression system has been modified to produce soluble human IL6 (hIL6) without TRX moiety in E. coli cytoplasm. A novel TRX gene fusion vector was developed that contained at the 3'-end of TRX gene a short DNA sequences encoding a linker peptide '-GSGSGVSQNYPIVQHHHHHH-', serving not only as a specific HIV-1 protease site but also providing six contiguous histidine (His) residues to foreign proteins. The cDNA for hIL6 was cloned into this vector resulting in plasmid pTRX@HISIL6. The cDNA for the HIV-1 protease has been cloned into another compatible plasmid pHMM2, resulting in plasmid pHMM2-PR. Both plasmids were transformed into E. coli strain GI724, and when induced for expression of both proteins, the correct processing of TRX@HISIL6 was obtained, producing hIL6 with His6-tag at the N terminus named HISIL6. A fraction of HISIL6 was found in soluble form and could be purified to homogeneity by Ni-NTA Superflow and ion-exchange chromatography. The biological activity of purified HISIL6 was measured by MTT method in an IL-6-dependent cell line 7TD1 to be 2.1 x 10(8) unit/mg.     

Expression of zonula occludens and adherens junctional proteins in human venous and arterial endothelial cells: role of occludin in endothelial solute barriers

ColE1-derived plasmids containing different recombinant genes which are controlled by the tac promoter were amplified following induction with IPTG, but no amplification occurred if product formation was not induced. The plasmid copy number of recombinant E. coli increased three- to sixfold within a period of about 6 h in shake flask experiments, batch cultures, and glucose-limited fed-batch cultivations. Plasmid amplification occurred in E. coli B strains as well as in K-12 strains with different plasmids (rop+ and rop-) coding for various heterologous proteins. The amplification was not caused by a toxic effect of IPTG, but was related to a strong inhibition of translation and chromosomal replication after the induction of heterologous gene expression. Similar to the amplification after chloramphenicol addition, plasmid replication proceeded even if oriC replication and translation were inhibited following strong induction of a recombinant gene. In accordance with the effect of chloramphenicol, the level of ppGpp, which is a negative regulator of ColE1 derived plasmid replication, decreased after induction.     

Subcellular localization and topology of the K88 usher FaeD in Escherichia coli

The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole cells by using isopycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X-100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the subcloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent-resistant form. Agglutination experiments with E. coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD-PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immunoblotting. Most of these fusion sites were located in the amino-terminal and carboxyl-terminal regions of FaeD. Six amino-terminal fusion proteins were soluble proteins in the periplasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane-bound FaeD-PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane-spanning segments and two relatively large periplasmic regions, at the amino-terminal and carboxyl-terminal end of the protein, respectively.     

Purification of gamma-crystallin from human lenses by acetone precipitation method

PURPOSE: The aim of this study was to develop a new purification method for human lens gamma-crystallin by utilizing its unique property of remaining soluble during acetone precipitation of water soluble (WS) proteins. METHODS: The WS protein fractions from lenses of donors of different ages were precipitated with 50% acetone (v/v) and the supernatant and precipitated protein fractions were collected following centrifugation. Among lens crystallins, gamma-crystallin remained soluble (recovered in the supernatant following centrifugation) while other crystallins were precipitated. To determine the recovery of maximal levels of gamma-crystallin as soluble protein during acetone precipitation, the WS proteins were precipitated under different conditions, and both supernatant and precipitated fractions were quantified for proteins and analyzed by size-exclusion chromatographic and Western blot methods. Based on these results, a three-step purification procedure for gamma-crystallin was developed which consisted of acetone precipitation followed by preparative isoelectric focusing (IEF) and size-exclusion HPLC of the soluble fraction. RESULTS: During precipitation of WS proteins by 50% (v/v) acetone, only gamma-crystallin remained soluble. The identity of gamma-crystallin was based on its Mr of 20 kDa on SDS-PAGE, co-elution with lens homogenate gamma-crystallin during a size-exclusion Agarose chromatography, immunoreactivity with anti-gamma-crystallin antibody on a Western blot and an overlap of its partial N-terminal sequence with gammaC-crystallin. A three-step procedure, as described above, provided a highly purified preparation of gammaC-crystallin from the WS protein fraction. The three-step procedure was also utilized to recover a highly purified human lens recombinant gammaD-crystallin preparation from E. coli lysate. CONCLUSIONS: The unique property of human lens gamma-crystallin of remaining soluble during acetone precipitation can be utilized to purify this crystallin by a three-step procedure. This procedure is also applicable in the purification of recombinant gammaD-crystallin from E. coli lysate.     

Hepatitis C virus E2 protein purified from mammalian cells is frequently recognized by E2-specific antibodies in patient sera

The envelope protein of hepatitis C virus (HCV) is composed of two membrane-associated glycoproteins, E1 and E2. To obtain HCV E2 protein as a secretory form at a high level, we constructed a recombinant chinese hamster ovary (CHO) cell line expressing a C-terminal truncated E2 (E2t) fused to human growth hormone (hGH), CHO/hGHE2t. The hGHE2t fusion protein was purified from the culture supernatant using anti-hGH mAb affinity chromatography at approximately 80% purity. The purified hGHE2t protein appeared to be assembled into oligomers linked by intermolecular disulfide bond(s) when density gradient centrifugation and SDS-polyacrylamide gel electrophoresis were employed. When the purified fusion protein was used for testing its ability to bind to antibodies specific for HCV by enzyme-linked immunosorbent assay, the protein was recognized by antibodies in sera from 90% of HCV-positive patients. Treatment of hGHE2t protein by beta-mercaptoethanol, but not by heat and SDS, significantly reduced its reactivity to the antibodies of patient sera, suggesting that intermolecular and/or intramolecular disulfide bonds are important for its ability to recognize its specific antibody and that the E2 protein contains discontinuous antigenic epitope(s).     

GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.     

Production and secretion of recombinant proteins in Dictyostelium discoideum

We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection.     

Purification of the putative coxsackievirus B receptor from HeLa cells

Human macrophage colony stimulating factor (M-CSF) has been successfully overexpressed in Escherichia coli AD494 (DE3) with an expression level of approximate 26% of the total cellular proteins. The truncated human M-CSF gene encoding the amino-terminal 149 amino acids was subcloned into the prokaryotic expression vector pET11d under the control of the inducible T7 promoter. Nearly 40% of the recombinant protein was in the soluble fraction which showed obvious stimulating effects on mouse macrophage colony formation and had an M-CSF specific activity of approximately 1 x 10(6) units/mg soluble protein.     

Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli. Lack of phosphorylation by protein kinase C

The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase. The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE. The yield was about 10 mg fusion protein/l lysate. After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1. The molecular mass of purified protein by SDS/PAGE was M(r) 43,000. The activity was inactivated by p-hydroxymercuribenzoate. The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein. In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C.