Recombinant expression of rat histidine decarboxylase: generation of antibodies useful for Western blot analysis

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Abstract

Histidine decarboxylase catalyses the formation of histamine, an important biological messenger. In spite of the essential biological functions exerted by histamine the knowledge about the mechanisms involved in the regulation of histidine decarboxylase is rather limited. This is most likely due to the limited supply of suitable tools, including highly specific antibodies. In the present study we describe the production and characterisation of specific antisera against rat histidine decarboxylase using recombinant protein synthesised in a bacterial expression system. The antisera were shown to effectively immunoprecipitate histidine decarboxylase activity in extracts of fetal rat liver as well as to detect the histidine decarboxylase protein by Western blot analysis of COS-7 cells expressing recombinant rat histidine decarboxylase. The results demonstrate the successful production of highly specific antisera to histidine decarboxylase which may become valuable tools in future studies of the structure and function of this enzyme.

Introduction

Histamine is an important biological messenger that can be found in a variety of tissues in the body. It fulfils important functions in several physiological and pathophysiological processes 1, 11, 17, 18. In the gastric mucosa, histamine has been shown to be a strong stimulator of acid secretion17, 18and specific histamine receptor antagonists have been developed which can be used successfully in the treatment of gastric ulcer2, 3. The enterochromaffin-like cells in the gastric mucosa have been identified as the major site of histamine production[13]. Although, the release of histamine from these cells has been shown to be strongly regulated by gastrin the exact mechanisms behind this regulation is not yet fully understood[13]. Histamine has furthermore been defined as a neurotransmitter4, 20, 25. The histaminergic neurons have their cell bodies located in the tuberomammillary nucleus with projections into almost all parts of the brain[31]. The histaminergic neurosystem is believed to regulate different activities in the brain like feeding, drinking, analgesia and sexual behaviour[31]. In addition to having functions in the gastric acid secretion and neurotransmission, histamine is a well known biological activator in the inflammatory process[11].

The biosynthesis of histamine is catalysed by the enzyme histidine decarboxylase (HDC; EC 4.1.1.22). In spite of the important biological functions that histamine exerts[18], very little is known about the mechanisms involved in the regulation of HDC[12]. The reason for this may be the limited supply of suitable tools, like specific antibodies that can be used for Western blot analysis as well as immunohistochemical detection of this enzyme. The specific activity of the enzyme is very low in most tissues. High HDC activity has been found in gastric mucosa[16], fetal rat liver[15], mast cells[24]and female mouse kidney[23]. Taguchi et al.[28]were able to purify HDC from fetal rat liver to homogeneity. Although the fetal rat liver was the richest known source of mammalian HDC, an eight step purification procedure resulted in a yield of less than 1 mg of 50% pure protein from 400 g of starting material[28]. Further purification, which gave rise to a homogenous protein, markedly inactivated the enzyme[28]. Nevertheless, an HDC antiserum useful for the immunohistochemical detection of the en zyme was raised against the homogenous protein28, 29, 32, 35. Unfortunately, the available amount of this antiserum is extremely limited. Thus, there are so far no information on its use in Western blot analysis of the protein.

A cDNA encoding mammalian HDC has been cloned from fetal rat liver[14]. In the present report we describe the expression and purification of mg quantities of this HDC in a bacterial expression system. Using the purified HDC several different antisera were raised and characterised for their usefulness in assays like Western blot analysis of the enzyme protein. The results demonstrate that the antisera obtained against mammalian HDC may become valuable tools for further investigations of the enzyme.

Section snippets

Materials

Alkaline phosphatase conjugated goat anti rabbit IgG, BCIP/NBT colour development reagents and pre-stained protein molecular weight standards were purchased from Bio-Rad (Stockholm, Sweden). Alkaline phosphatase conjugated goat anti guinea-pig IgG and bacterial protein A adsorbent were bought from Sigma (Stockholm, Sweden). l-[carboxyl-14C]Histidine (46.5 mCi/mmol) was obtained from New England Nuclear (Stockholm, Sweden). QuickChange™ Site-Directed Mutagenesis Kit and pBluescript II KS+ were

Expression of recombinant HDC in E. coli

The cDNA used in the present study, encoding fetal rat liver HDC, was originally isolated and provided by Joseph and colleagues[14]. The cDNA encodes a protein with a molecular mass of about 74 kDa. To express the recombinant HDC to high levels in E. coli we used the pET His·Tag™ system. In this system target genes are placed under the control of a strong T7 promoter and induction is achieved in an E. coli strain (BL21(DE3)) producing T7 RNA polymerase upon incubation with IPTG7, 27. The

Discussion

The purification of HDC to homogeneity from various mammalian tissues have been shown to be associated with great difficulties. A major problem is the lability of the purified protein which gives rise to a rapid loss of enzyme activity8, 33, 34. Furthermore, HDC is present in very low amounts even in tissues containing high HDC activity. Thus, in most cases the final amount of pure HDC protein was extremely small33, 34. The only exception from this was the purification of HDC from fetal rat

Acknowledgements

This study was supported by grants from the Swedish Cancer Society and the Albert Påhlsson, the Crafoord and the Wiberg foundations.

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