Expression of Human Placental Alkaline Phosphatase in Escherichia coli

doi:10.1006/prep.1994.1030

Protein Expression and Purification

Volume 5, Issue 2, April 1994, Pages 192-197

https://doi.org/10.1006/prep.1994.1030 Get rights and content

Abstract

The human placental alkaline phosphatase gene was subcloned for expression in Escherichia coli. A shortened form of the alkaline phosphatase, lacking 29 C-terminal amino acids which constitute a membrane anchoring domain, is expressed to about 5% of total protein. Most of the enzyme is present in an insoluble form; however, soluble enzyme can be detected in Western blots and activity tests. The protein is located in the periplasm since its signal peptide is cleaved off. Like the wild-type enzyme, recombinant, shortened placental alkaline phosphatase is inhibited by L-phenylalanine. The amount of active enzyme can be increased by addition of magnesium but not zinc to the medium. Using optimized fermentation conditions, 11 kU of soluble and active alkaline phosphatase are produced per liter. Coexpression of potential folding aids like peptidyl-prolyl cis-trans isomerase or disulfide isomerase does not lead to an increase of soluble enzyme, either when overexpressed separately or from an operon.

References (0)

Cited by (21)

Development of recombinant secondary antibody mimics (rSAMs) for immunoassays through genetic fusion of monomeric alkaline phosphatase with antibody binders
2023, International Journal of Biological Macromolecules
In conventional immunoassays, a secondary antibody is used to amplify the signal generated by the binding of the primary antibody to the target analyte. Due to concerns regarding animal use and cost-inefficiency of secondary antibody productions, there is a significant demand for the development of recombinant secondary antibody mimics (rSAMs). Here, we developed rSAMs using a signal-generating enzyme, monomeric alkaline phosphatase (mALP), and antibody-binders, including monomeric streptavidin (mSA2) and mouse IgG1- or rabbit IgG-binding nanobodies (MG1Nb or RNb). The mALP-MG1Nb, mALP-RNb, and mALP-mSA2 were genetically constructed and produced in large quantities using bacterial overexpression systems, which reduced manufacturing costs and time without the use of animals. Each rSAM exhibited high and selective binding to its respective primary antibody, generating linear band signals corresponding to the amounts of target analytes in western blots. The rSAMs also successfully generated sigmoidal signal curves that increased as the sample concentration increased. Moreover, they generated stronger signals than conventional ALP-conjugated secondary antibodies and SA, particularly in the medium to high sample concentration range, in both indirect and sandwich-type indirect ELISAs at the same sample concentration. The rSAMs we developed here may provide new insights to develop novel immunoassay-based analytical and diagnostic tools.
A simple whole cell microbial biosensors to monitor soil pollution
2017, New Pesticides and Soil Sensors
Three-quarters of the earth’s surface is covered by water and the rest of it by soil. Soil contains a combination of minerals, organic matter, gases, and countless numbers of organisms. Over the last few years, soil has been contaminated by the accumulation of anthropogenic wastes, for the most part. These contaminants reduce soil fertility, increase toxic dust, and change soil structure. To limit the impact of these on human health and the environment, there is an urgent need for simple and cost-effective quantitative sensors to detect soil pollution. The most commonly available methods for determining contaminants in soil are gas chromatography, atomic absorption spectroscopy, inductively coupled plasma-atomic emission/mass spectroscopy, and liquid chromatography. However, these methods are laborious, time-consuming, and expensive. The best alternative for assessing soil pollution is, instead, a “bacterial biosensor.” Biosensors are analytical devices, coupling a biological sensing element (promoter) with a reporter to produce a signal that is proportional to the analyte concentration. Expression of these reporter signals is easily traceable, thereby superseding the cost-effectiveness of other sophisticated instruments. The chapter explores the recent advancements in bacterial biosensors and their implications for soil pollution management.
Functional expression, purification, and characterization of the extra stable human placental alkaline phosphatase in the Pichia pastoris system
2004, Protein Expression and Purification
Human placental alkaline phosphatase was successfully cloned in the yeast system Pichia pastoris. The recombinant enzyme was over-expressed as a secreted protein in the cultured medium. The enzyme was extremely stable, which resulted in a total recovery of the enzyme activity after the purification process. The purified enzyme preparation was apparently homogeneous as examined by the polyacrylamide gel electrophoresis, analytical gel-permeation chromatography, and analytical ultracentrifugation. The final enzyme preparation showed a purification of 803-fold from the culture medium with a specific activity of 578 U/mg of protein. Fluorescence spectroscopic analyses showed multiple unfolding steps in the urea denaturation process of the homodimeric recombinant enzyme. Extensive conformational change of the enzyme in urea was detected by the analytical ultracentrifugation and the size-exclusive chromatography. The quaternary structure of the enzyme is quite stable. No indication of dissociation was observed after extensive tertiary structural changes.
Overproduction of Bacterial Protein Disulfide Isomerase (DsbC) and Its Modulator (DsbD) Markedly Enhances Periplasmic Production of Human Nerve Growth Factor in Escherichia coli
2001, Journal of Biological Chemistry
Citation Excerpt :
Consistent with this proposal, expression of a bovine pancreatic trypsin inhibitor containing three disulfide bonds (nonconsecutive-type) yielded intermediates mostly with aberrant disulfide bonds in a dsbD deletion mutant (14). Similarly, the yield of active human placental alkaline phosphatase was low, whereas bacterial alkaline phosphatase was normally produced in thedipZ (dsbD) mutant presumably because of formation of aberrant disulfide bonds (39). It thus seems clear that DsbD, as well as DsbC, can become overloaded upon overexpression of heterologous proteins having multiple disulfide bonds.
Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor β (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permitdsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.
Human placental alkaline phosphatase: Expression in Pichia pastoris, purification and characterization of the enzyme
1998, Protein Expression and Purification
The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeastPichia pastorisand the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pIrange of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems.
Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli
1995, Current Opinion in Biotechnology
Interest continues to increase in the use of folding modulators to overcome problems with heterologous protein folding in Escherichia coli. Currently, this approach, though highly successful with a number of individual proteins, remains a somewhat hit-and-miss affair. Ongoing research directed at unraveling the precise role and specificity of these folding modulators should generate a clearer understanding of the potential and limitations of overexpressing folding catalysts in vivo. This will facilitate the development, in the not too distant future, of a more structured and rational approach to improving the folding of heterologous gene products in E. coli.

View all citing articles on Scopus

View full text

Regular ArticleExpression of Human Placental Alkaline Phosphatase in Escherichia coli

Abstract

Regular Article
Expression of Human Placental Alkaline Phosphatase in Escherichia coli