The variability of extracts derived from Alternaria (ALT) is well documented. One possible solution to this problem might be the production of recombinant allergenic proteins in E. coli by inserting DNA taken from ALT which is specific for production of a particular allergen. To explore this possibility, the following experiments were performed.
A strain of ALT was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration for 5 days. Rapidly expanding mycelia were removed from the culture and frozen in liquid nitrogen. Frozen mycelia were ground in liquid nitrogen and disrupted with short bursts in a high speed homogenizer. Homogenized mycelia were denatured in guanidine thiocynate in the presence of RNase inhibitors and extracted using a phenol chloroform method. Purified RNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification.
Amplified DNA as well as whole cDNA was modified to contain a EcoR1 restriction site on one side and a Not1 site on the other end. Modified DNA was then spliced into lambda gt11 phage. The packing reaction yielded recombinant libraries with phage titers in the range of 10E5 plaque forming units. Amplified libraries were screened for the presence of allergenic proteins with monoclonal antibodies (MAb) directed to ALT allergens and with IgE containing human sera from ALT sensitive patients. Positive plaques were cloned and used in the production of E. coli lysogens. Positive colonies were chosen and grown under conditions specific for the production of the recombinant protein. Expressed proteins were tested using a MAb based sandwich ELISA for allergenic proteins from Alternaria and by SDS-PAGE immunoblotting analysis.
Further analysis of these recombinant proteins should permit a better understanding of ALT allergens.
3. STRUCTURAL DETERMINATION OF A HUMAN IGE RECOGNIZED PROTEIN FROM
ALTERNARIA ALTERNATA. C.Barnes, D.Rosenthal, F.Pacheco, J.Landuyt and
J.Portnoy. ChildrenÕa Mercy Hospital and UMKC, Medical School, Kansas
City, MO 64108
5. PARTIAL AMINO ACID SEQUENCE FOR THE ALT AI ALLERGEN. J Portnoy MD,F
Pacheco MS, D Rosenthal,J Landuyt BS, C Barnes PhD. Kansas City,
Missouri.
6. PRODUCTION OF A FUSION PROTEIN CONTAINING RECOMBINANT ALT AI, A MAJOR
ALTERNARIA ALLERGEN. F Pacheco MS, D Rosenthal, J Portnoy MD,, J Landuyt
BS, C Barnes PhD. Kansas City, Missouri.
7. STUDIES ON THE MRNA FOR ALTERNARIA ALLERGEN ALT AI BY POLYMERASE
CHAIN REACTION (PCR). C Barnes PhD , D Rosenthal, F Pacheco MS, J Landuyt
BS, J Portnoy MD. Kansas City, Missouri.
9. NORTHERN BLOTTING STUDIES ON ALT AI MRNA AND 5.8S RRNA IN NINE STRAINS
OF ALTERNARIA. D Rosenthal, R Crenshaw MS, R Esch PhD, F Pacheco MS, J
Landuyt BS, F Hu DVM, C Barnes PhD, J Portnoy MD. Kansas City, Missouri
and Lenoir, North Carolina.
10. IGE BINDING TO A SYNTHETIC RECOMBINANT PEPTIDE CONTAINING THE N-
TERMINAL AA SEQUENCE OF ALT A1. F Hu DVM , F Pacheco MS, J Landuyt BS, D
Rosenthal, C Barnes PhD,.J Portnoy MD. Kansas City, Missouri.
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IgE recognized proteins derived from environmental sources cause
significant human morbidity and mortality. Elucidation of structural
characteristics of these proteins contributes to improved diagnosis and
treatment of asthma and allergy. To study the structure of a 31kDa
allergenic protein identified in the mold, Alternaria alternata (ALT), the
following experiments were performed.
ALT was grown in a fermenter with constant stirring and aeration.
Monoclonal antibodies (MABÕs) against the 31kDa protein were produced from
electrophoretically separated material. Additional protein was purified by
MAB affinity chromatography. For RNA purification, rapidly expanding
mycelia was ground in liquid nitrogen and extracted using a phenol
chloroform method. RNA was reacted with reverse transcriptase and first
copy ssDNA was amplified by PCR. Amplified DNA as well as ds-cDNA was
inserted directionally into phage and a cDNA library was produced. The
library was screened with MABÕs and IgE containing human sera from ALT
sensitive patients. Positive clones were analyzed by PCR and sequenced.
PCR analysis of cDNA using an oligonucleotide derived from the N-terminal
sequence and oligo dT indicates a nucleotide of 295 base pairs. Sequence
analysis of the PCR product yields a reading frame containing 66 amino
acids. A computer search for this sequence found no homologous proteins.
Northern blotting analysis indicates the PCR product to be the C-terminal
third of a 1000 base mRNA segment.1994 Abstracts
4. IDENTIFICATION OF THE ALT AI ALLERGEN IN EIGHT STRAINS OF
ALTERNARIA.
D Rosenthal, R Crenshaw MS, R Esch PhD, F Pacheco MS, J Landuyt BS, C
Barnes PhD, J Portnoy MD. Kansas City, Missouri and Lenoir, North
Carolina.
The variability of extracts derived from Alternaria (ALT) is well
documented. At the protein level significant allergens are often absent
or in very low concentrations. To to determine if these variations occur
at the DNA level, the following experiments were performed.
Eight strains of ALT obtained from 4 different research groups were grown
on minimum salts and glucose in a fermentation container with constant
stirring and aeration for 5 days. Rapidly expanding mycelia were removed
from the culture and frozen in liquid nitrogen. Frozen mycelia were
ground in liquid nitrogen and disrupted with short bursts in a high speed
homogenizer. Homogenized mycelia were denatured in guanidine thiocynate
in the presence of RNase inhibitors and extracted using a phenol
chloroform method. After quantitative measurements were made by UV
absorption, alliquots containing equal amounts of RNA were applied to a
denaturing agarose gel, electrophoresed and blotted by capillary action
onto nitrocellulose. RNA blots were probed with a radiolabeled 300 BP DNA
fragment which codes for the N-terminal portion of the Alt aI allergen.
Probed blots were visualized on a phosphorimager.
Analysis of the RNA blot showed a single band at about 1000 bases in each
of the eight strains of Alternaria tested. Densitometric analysis of the
blots obtained indicated that some strains contained as much as 10 times
the mRNA for this allergen as other strians. The results of the northern
blot analysis is compared with quantitative ELISA analysis for the
protein allergen in these same strains. These data suggest that
quantitative differences in allergen production among differing strains
of Alternaria occur not only at the protein level but also at the level
of the genetic material. Post translational processing of proteins is an
important contributor to the complexity of ALT extracts and that such
processing must be accounted for when standardization is attempted.
Sequencing of IgE recognized proteins either at the amino acid or DNA
level has been conducted for many significant allergens. Elucidation of
structural characteristics of these proteins contributes to improved
diagnosis and treatment of asthma and allergy. To study the structure of
a 31kD allergenic protein identified in the mold, Alternaria
alternata(ALT), the following experiments were performed.
ALT was grown in a fermenter with constant stirring and aeration.
Monoclonal antibodies (MAB's) against the 31kD protein were produced from
electrophoretically separated material. Additional protein was purified
by MAB affinity chromatography. For RNA purification, rapidly expanding
mycelia were ground in liquid nitrogen and extracted using a phenol
chloroform method. RNA was reacted with reverse transcriptase and first
copy ssDNA was amplified by PCR. Amplified DNA as well as ds-cDNA was
inserted directionally into phage and a cDNA library was produced. The
library was screened with MABÕs and IgE containing human sera from ALT
sensitive patients.
PCR analysis of cDNA using an oligonucleotide derived from the N-terminal
sequence and oligo dT indicates a nucleotide of 295 base pairs. Sequence
analysis of the PCR product yields a reading frame containing 85 amino
acids. A computer search for this sequence found no homologous proteins.
Northern blotting analysis indicates the PCR product to be the C-terminal
third of a 1000 base mRNA segment.
The allergenic composition of Alternaria (ALT) extracts is extremely
variable. This makes it difficult to characterize specific allergenic
glycoproteins. One solution to this may be with the production of
recombinant allergenic proteins. By inserting DNA coding for a known ALT
allergen into an appropriate vector, it is possible to produce usable
quantities of protein, To this end, the following experiments were
performed.
A strain of ALT was grown on minimum salts and glucose in a fermenter
with constant stirring and aeration for 5 days. Rapidly expanding mycelia
were removed from the culture and frozen in liquid nitrogen. These were
next ground in liquid nitrogen and RNA was extracted using a phenol
chloroform method. RNA was reacted with reverse transcriptase and an
aliquot of first copy single strand DNA was enriched for the presence of
cDNA coding for an Alternaria allergen by PCR amplification. cDNA
libraries were screened for the presence the desired insert by PCR and
for allergenic proteins with monoclonal antibodies (MAb) directed to ALT
allergens and with IgE containing human sera from ALT-sensitive patients.
Positive plaques were cloned and used in the production of the desired
DNA. Specific oligonucleotide primers were designed to give a PCR product
containing a full copy of the desired DNA along with the necessary
restriction sites to allow insertion into a plasmid specific for
expresson of protein.
Colonies were screened for expressed proteins by SDS-PAGE and positive
clones were sequenced. The product was fused to the C-terminus of
glutathione sulfhydryl transferase which aided in purification and could
be easily removed enzymatically. Expressed proteins were tested using a
MAb-based sandwich ELISA for allergenic proteins from Alternaria and by
SDS-PAGE immunoblotting analysis. Further analysis of this product should
clarify many of the properties of Alt aI
Traditional protein isolation and purification methods including
immunoblotting have yielded less than a consensus among researchers about
the nature of the allergens found in Alternaria (ALT) extracts. Because
of the apparent differences in allergens at the protein level, efforts to
characterize allergen content in differing strains of ALT must
concentrate on the level of the nucleotides. PCR is a powerful tool for
the analysis of native DNA and cDNA inserts cloned into E. coli through
the use of phage or plasmid vectors. To characterize ALT allergen content
at the DNA and mRNA levels, the following studies were conducted.
ALT was grown on defined medium in a fermenter with constant stirring and
aeration for 5 days. Rapidly expanding mycelia were removed, frozen and
then ground in liquid nitrogen, and disintegrated in a high speed
homogenizer. The product was denatured in guanidine HCl with RNase
inhibitors and extracted using a phenol chloroform method. RNA was
reacted with reverse transcriptase. DNA was then spliced directionally
into lambda gt11 phage. Amplified libraries were screened for the
presence of DNA coding for allergenic proteins with PCR using synthetic
oligonucleotides specific for a 150 base pair region of Alt aI. Positive
plaques were then screened for size using PCR with oligonucleotides
residing on either side of the insert region.
PCR screening of cDNA libraries indicates that mRNA coding for Alt a1 is
as large as 600 base pairs. MRNA and DNA specific for the Alt aI allergen
can be identified from several strains of ALT using PCR techniques. 1995 Abstracts
8. PRODUCTION OF A RECOMBINANT PROTEIN FROM ALTERNARIA CONTAINING THE
REPORTED N-TERMINAL OF THE ALT A1 ALLERGEN. CS Barnes, F Pacheco, J
Landuyt, D Rosenthal, F Hu , J Portnoy . Section of Allergy/Immunology,
The Children's Mercy Hospital, Kansas City, MO.
Allergen content of extract derived from Alternaria is somewhat
variable. Allergenic molecules from Alternaria that appear as differing
molecular size bands on IgE probed immunoblots may have a great deal of
sequence homology and differ only in the length of the amino acid chain.
One method to study this problem is to produce recombinant proteins from
Alternaria. To explore these possibilities, the following experiments were
performed.
A strain of Alternaria was grown on minimum salts and glucose in a
fermentation container with constant stirring and aeration. Rapidly
expanding mycelia were removed from the culture and mRNA was extracted.
Purified mRNA was enriched for the presence of DNA coding for an
Alternaria allergen by PCR amplification. Modified DNA was then spliced
into lambda gt11 phage and yielded a recombinant library with 10E5 PFU. The
library was screened for the presence of allergenic proteins using IgE
containing human sera from Alternaria-sensitive patients. Positive plaques
were cloned.
PCR analysis of positive clones using an oligonucleotide from the
reported N-terminal sequence of Alt a1 indicated an insert of 295 base
pairs. Sequence analysis yielded a reading frame containing 84 amino acids
and confirmed that this segment contained the code for the reported N-
terminal amino acid sequence of Alt a1. A computer search for this
sequence found no homologous proteins in the Entrez¨ sequences. Northern
blotting studies on RNA purified from nine strains of Alternaria with the
radiolabeled 274 BP DNA fragment indicated that this sequence was present
in all strains. The 274 BP nucleotide was spliced into the Pflag¨ vector
and clones containing insert in the proper reading frame were identified.
The presence of recombinant protein in the clones was verified by SDSPAGE
time studies. Protein produced in time studies was shown by immunoblotting
and sandwich EIA to bind human IgE from Alternaria sensitive patients.
This recombinant protein, containing amino acid sequence for Alt a1, is
bound by human IgE and therefore should be useful as a model for studying
allergy to the native Alternaria glycoprotein. Further research should
define where this sequence occurs in the Alternaria genome and should
determine the sequence of the entire protein.
Allergen content of extracts derived from Alternaria (ALT) varies greatly.
To determine if similar variations are present in mRNA we performed the
following experiments.
For 9 strains of ALT, rapidly expanding mycelia were frozen at -70¡C,
homogenized, denatured in guanidium thiocynate in the presence of RNase
inhibitors, and extracted using a phenol chloroform method. Aliquots
containing equal amounts of RNA were applied to a formaldehyde denaturing
agarose gel, electrophoresed, and blotted by capillary action onto nylon66
membrane. RNA blots were probed with a radiolabeled 274 BP DNA fragment
which codes for the reported N-terminal portion of Alt aI and with a 156
BP DNA fragment from 5.8S ribosomal RNA. Spent culture filtrate and
extracted cellular material were assayed for the presence of Alt a1 by
sandwich EIA.
When normalized using the 5.8S rRNA, Alt a1 mRNA in the 9 strains varied
as much as 4-fold. Alt a1 protein correlated only moderately (.56, p=0.1)
with the amount of mRNA. This correlation was much lower (.10) for another
ALT allergen, GP70.
The observed low correlation between mRNA and allergen levels may be
caused by protein degradation or immunologically cross reactive
proteins.
The characteristics of Alternaria (ALT) allergens may be studied via
production of recombinant allergenic proteins. Inserting DNA coding for a
known ALT allergen into an appropriate vector allows the production of
usable quantities of the relevant allergen devoid of carbohydrate. The IgE
binding characteristics of this allergen devoid of its carbohydrate moity
will provide a great deal of insight into the nature of the allergen. To
begin this process of discovery, the following experiments were performed.
A strain of ALT was grown on minimum salts and glucose in a fermenter with
constant stirring and aeration for 5 days. Rapidly expanding mycelia were
removed from the culture and used to produce a cDNA library for
Alternaria.The library was screened for allergenic proteins with
monoclonal antibodies (MAb) directed to ALT allergens and with IgE
containing human sera from ALT-sensitive patients.A clone of E. coli
containing 300 BP segment of Alternaria DNA was identified. Sequence
analysis determined that this segment contained the code for the known N-
terminal amino acid sequence of the Alt a1 allergen. The segment was
spliced into the Pflag¨ vector for protein production and 4 clones
containing the insert in the proper reading frame were identified. These
clones were then induced to produce the recombinant protein. Recombinant
proteins produced were purified by affinity chromatography.
Expressed proteins were tested for IgE binding by SDS-PAGE immunoblotting
analysis using sera from Alt sensitive patients and by three ELISA based
methods. Further analysis of this product should clarify many of the
properties of Alt aI.
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