.BETA.-HEXOSAMINIDASE, SEQUENCE ADN DE CILIES LA CODANT ET SON UTILISATION

.BETA.-HEXOSAMINIDASE, DNA SEQUENCE FROM CILIATES FOR CODING THE SAME AND USE THEREOF

Abrégé français

L'invention concerne un acide nucléique codant pour une beta -hexosaminidase de ciliés.

Abrégé anglais

A nucleic acid coding for a .beta.-hexosaminidase from clliates.

Revendications

CLAIMS:
1. Use of a nucleic acid or parts thereof coding for a .beta.-hexosaminidase
from
ciliates for the homologous or heterologous expression of recombinant pro-
teins and peptides, and for homologous or heterologous recombination
("knock-out, "gene replacement").
2. The use according to claim 1, characterized in that said nucleic acid codes
for an extracellular .beta.-hexosaminidase.
3. The use according to claim 1 or 2, characterized in that said nucleic acid
has
the sequence shown in Figure 1, especially that of Seq. Id. No. 1.
4. The use according to claims 1 to 3, wherein said nucleic acid codes for an
N-terminal fragment of a protein having the sequence MQKILLITFLLGIALAQ.
5. Use of a nucleic acid according to any of claims 1 to 4 in a method in
which
said nucleic acid is combined with the usual, in homologous or heterologous
expression, enhancers, promoters, operators, origins, terminators, antibiotic
resistances, or other nucleic acids or DNA fragments, or sequences of any
kind from viroids, viruses, bacteria, archezoans, protozoans, fungi, plants,
animals or humans.
6. The use according to claim 5 in a method in which the nucleic acid is
incorporated or inserted into a vector, a plasmid, a cosmid, a chromosome
or minichromosome, a transposon, an IS element, an rDNA, or any other
kind of circular or linear DNA or RNA,
7. A peptide having the sequence MQKILLITFLLGIALAQ.
8. A nucleic acid coding for the peptide according to claim 7.
9. Use of the peptide according to claim 7 or of the nucleic acid according to
claim 8 for the homologous or heterologous expression of recombinant pro-

-2-
teins and peptides, and for homologous or heterologous recombination
("knock-out, "gene replacement").

Description

CA 02372285 2002-O1-14
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(~iexosaminil,~iase and a ONA Seoyence_Codina it
The present invention relates to a nudelc acid coding for a ~-hexosaminidase.
The expression of foreign proteins in microorganisms, such as bacteria, yeasts
or
mammal cells, is of great importance to the biotechnological proparation and
production of recombinant proteins. Thus, bacterial expression systems based
on
E, coil or B. subti!!s are used for the production of recombinant peptides or
proteins, such as insulin, interleukin-2, tissue plasmlnogen activator,
protea~s
and Iipases. In Gram-negative bacteria, the expression systems are mostly
based
on the use of genetic elements such as the lac operon or the tryptophan
operon.
The proteins foreign to the host are produced either into "inclusion bodies"
wiri~in
the cell, or when expression systems based on (i-lactamase genes are used,
into
the peHplasmic space. The production of recombinant proteins into the
surrounding
fermentation medium has not been established. In Grem-Positive bacteria, to
date,
almost exdusiveiy cell-inherent proteins are introduced and expressed.
Yeasts, such as S. aenvislse, Kluyveromyc~s larctis or PichJa pastorfs, are
also
employed for the heterologous expression of recombinant proteins, such as
human
factor XIIIa, bovine pro-chymosin, or surtace antigens. In yeasts, the
expression
systems are based on shuttle vectors (vectors having both yeast and bacterial
porh~ons) which are constructed from the genetic elements of gaiacko-kinase-
epimerase, acid phosphatase or aicohvl-dehydrogenase. As a rule, the recombi-
nant protein is produced Into the cytoplasm of the cell. When yeast-inherent
signal
sequences, such as the alpha factor, are used, the expressed proteins may also
be
secreted into the fermentation medium. The giycosylation of secreted proteins
is
effected according to the "high mannose" type.
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Mammal cells, such as various tail types from rodents (CNO cells, C127 cells)
or
simians (vero, CV-1 or COS cells), are also employed for the heterologous
expres-
-- sion of recombinant proteins. Nere, the expression systems are based on
recombi-
pant viruses (BPV vector) or on shuttle vectors. To regulate the expression,
viral
SV40 enhancer/promoter systems or cellular enhancer elements are employed.
The recombinant proteins, such as erythropoietin, are secreted into the
fermenta-
tion medium because the foreign gents usually bring their own signal
sequences,
which are understood by the expression system and used for targeting.
Further, for the biotechnological production of glycosylated extrecNlular
enrymes,
ciliates such as Tetrahymena are employed. Ciliates will grow on inexpensive
fermen, tattoo media using standard fermentation methods. For the
transformation
of such ciliates, vectors are available which are based on the rDNA elements
of the
,,
ciliate Tetrahyme»a. For the heterologous expression of bacterial proteins in
ciliates, DNA ~nstrucCs consisting of genes Ilrom Tetrahymena are employed,
When suitable genetic elements for the regulation of the transcrtpdon,
targeting
and glycosylation of tnrsign proteins are available, dilates are an Ideal
e~cpression
system far the inexpensive production of therapeutic recombinant proteins.
The Gram-negative bacterial expr~essian systems used to date usually lead to
the
formation of "inclusion bodlos" in the cell, accompanied by a denakuring of
the
proteins. To recover the recombinant protein, the cells must be lysed, and the
denatured inactive protein must be folded back to function. This causes
additional
cost-intensive process steps and reduces the yield of the desired protein.
Glycosy-
iatlon, which is important to eukaryotic proteins, is completely omitted. When
Gram-positive bacterial expression systems arse used, degradation of the
target
protein due to high proteolytic activities in the fermentation broth is an
additional
problem.
When yeasts are used for heterologous expression, the desired target protein
is
often produced into the cell only from where it must be removed by cell lysls.
As in
bacterial expression systems, this causes additional time- and cost-intensive
process steps. When yeast~inherent signal peptides are used, the foreign
proteins
are not correctly spliced and gtycosylated for secretion.
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In contrast, when mammal cell systems are employed for the production of
recombinant proteins, the desired proteins are fiound in the fermentation
medium
in an extracellular state, correctly spliced and glycosylated. However, what
Is
disadvantageous here is, on the one hand, the low expression rate due to the
defective processing and ineffident translation of genes which have been intro-
duced into the genome of the production cell line via viral vectors. On the
other
hand, the serum-containing fermentation media for mammal cells are extremely
cost-Intensive. In addition, the fermentation technology for the shear-
sensitive cell
lines is complicated and similarly expensive due to constnxtton for bubble-
free
aeration. Further problems arise from the high Infection risk for the cell
lines from
mycoplasmas and viruses. All in all, the use of mammal cells for the
biotechnviogl-
cal._preparatton of recombinant proteins results in very high costs, safety
demands
and low yields.
To the use of dilates, such as Tetrahymena, the above mentioned drawbacks In
the production of proteins do not apply. Thus, for example, some acid
hydrofases
which are involved in the digestion of food partides are exported from the
cell in
high quaMitles and with complex giycosylatlon.
In 3. Euk. Microblot. 43 (4), 1996, pages Z95 to 303, Alam et al. describe the
cloning of a gene which codes for the add a-glucosidase of Tetrahymena pyrl-
formis. However, only a small portion of the protein is exported from the
cell.
However, to date, it has not been possible to bring other foreign gtycosylated
eukaryotlc proteins to expression in dilates which are also secreted into the
fermentation medium. This is due to the fact that the DNA sequences from
cittate-
inherent secreted add hydrolases which are necessary for the oonstnrction of
expression vectors have not been known to date.
It has been the object of the invention to provide a DNA sequence 1br the
expres-
sion of secreted proteins of Gliates. The DNA sequence is to enable
heterolo9ous
proteins In an expression system to be exported into the fermentation medium
after transformation in ciliates. This system Is also to achieve a high
expression
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rate of the heterologous proCein, which is exported in large amounts from the
cNl
under culture conditions.
This object is achieved by a system in which a nucleic add having a sequence
with
Seq. Id. No. I and coding for pr-hexosamlnidase Is used.
In particular, the inventive DNA sequence of the ~-hexosaminldase includes a
signal peptide and a propeptide, and optionally further genetic elements for
the
targeting of proteins. The use of these sequences In a victor enables heterolo-
gously expressed proteins to be exported from tfie cell and thus to be
purffled from
the fermentation broth without a cell lysis.
Figure 1 shows a nucleic add coding for p-hexosamtnidase from dilates. Figure
2
shows a corresponding expression product of the nucleic acid according to Seq.
Id.
No. 1. The invention also relates to this protein according to Seq. Id. No. Z.
In particular, the invention relates to the signal sequence of the protein
dccordtng
to the invention. This is preferably amino adds 1 to 17 of the protein
according to
the invention. The invention also nlstes'to a nucleic add which codes for the
N-
terminal fragment. This is preferably a fragment of the nucleic acids
according to
the invention, especially with the nucleic acid sequence from 1 to 51
according to
Figure 1.
A further aspect of the invention Is the use of a nucleic add sequence of acid
hydrolases according to the invention or parts thereof far the homologous or
heterologous expression of recombinant proteins and peptides, and for homolo-
gous or heterologous recombination ("knock-out, "gene replacement").
The invention also relates to a method in which the nucleic add or parts
thereof
according to the invention which code for p-hwcosamtnldase is combined with
the
usual, in homologous or heteroloQous expression, anhancers, promoters, opera-
tors, origins, terminators, antibiotic resistances, or other nucleic adds or
DNA
fragments, or sequences of any kind from viroids, viruses, bacteria,
archezoans,
protoZOans, fungi, plants, animals or humans.
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In particular, the nucleic acid or parts thereof according to the invention is
inserted
into a vector, a plasmid, a cosmid, a chromosome or mlnlchromosome, a transpo-
son, an IS element, an rDNA, or any kind of Circular or linear DNA or RNA,
The skilled person will understand that nucleic acids having at least
40°Aa homology
with the nucleic acid according to Seq. td. No. 1 can also be employed
according to
the invention. The protein according to Seq. Id. No. 2 can also be modiaqed
without
losing its function. Thus, for example, so-called conservative exchanges of
amino
acids may be performed. Thus, for example, hydrophobic amino acids can be
interchanged.
For the purification and Isolation of p-hexosaminidase from ciliates and 1br
deter-
mining its sequence, the following methods can be used. This is illustrated by
the
following Examples.
From a total of 3.21 of cell culture, cells of the ciliate Tirtrahyme»a in
late loga-
rithmic growth phase were washed into 400 ml of starving medium (10 rnNf Tris-
HCI, pH 7.4), and the cells were incubated for another 4 hours with shaking,
Then,
the cell-free culture supernatant was harvested and filtered through a number
of
filters with decreasing pore diameters to remove any particular material. The
filtrate was concxntrated with an Amicon ultraflltration cell and rebuffered
into the
starting buffer for the subsequent ion-exchange chromatography (IEC).
The collected chromatographic fractions were tested with specific 4-
nitrophenyl
substrates fior acid hydrolases, add phosphatase (aPAse) and p-hexosaminidase
(p-Hex), and the fractions having the highest p-Hex activity were combined.
They
were concentrated by another Amicon ultrafiltration and rebuffered Into the
starting buffer for affinity chromatography. The collected chromatographic
frac-
tions were tested for enzymatic activities as described above, and the
fractions
having the highest activity for an add hydrolase were combined and rebufPered
Into phosphate buffer (PB). Prom a total of eight purificatians, the
chromatographic
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fractions containing hydrolases were pooled, concentrated, portioned and
frozen
until further charactertzatlon.
Part of the thus purified hydrolase was separated by two-dtrnenstonal SDS gel
electrophoresis (a total of eight gels), the main spot was respectively
punched out
and collected. The protein present in these gel pieces, p-Hex, was digested
with
the protease trypsin. The thus obtained peptide fragments were leached out of
the
gel pieces and separated by reverse-phase HPl.C (RP-HPLC). The purity of the
HPLC fractions was tested with a mass-spectroscopical method (MAl.DI-MS), and
the pure fractions, i.e., those which contained only one peptide species of a
defined mass, were sequenced from their N-terminus using t_drnan degradation.
From the sequence of the peptide fragments obtained by the trypsln digestion,
ti-hexosaminidase-spedflc PCR primers were established. By a sequence compari-
son with the existing ~-Hex sequences, a preselection of the primer
combinations
for RT-PCR could be made. With this information, the first spedfic cDNA
fragments
were amplifted and sequenced, using RT-PCR, from isolated whole RNA whose
quality was previously tested by Northern hybridlzaGlon. Using these
fragments,
sequence-speciftc primers were constructed which were employed for further PCR
experiments. Each primer and each partial sequence obtained was checked by
data
base alignment, and prior to further experiments, it was also checked for any
overlooked vector sequences, inter alta. By 5'- and 3'-RACE, the cDNA sequence
was elongated at the 5'- and 3'-termini. The thus completed sequence of the (i-
Hex
cDNA then served as the basis for further sequence analyses.
The thus established sequence of a ~-hexosamtntdase from the ciliate reads as
set
forth in Figure 1. In total, this sequence comprises 1836 base pairs indudinq
5'-
and 3'-non-translated regions of p-hexosaminidase with an open reading trarne
having a IenQth of 165fi base pairs. The complementary amino add sequence has
a length of 551 amino acids and reads as set forth in Figure 2.
The sequence of the (3-htxosamtnldaae has a total of 9 glycosylation sites and
contains a signal peptide and a pro sequence for targeting the enzyme through
the
sorting mechanism of the cell.
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SLQUg112DROTOKOLL
<1i0> Hartmann, Marcus
<i20> 8aura Hydrolaaen Bowie diwae kodierenda DNA-Saquena auo
Ciliaten and deran Veswendung
c130> Flarcmann, Marcus
<140>
c141>
W ao> 3
c1~0~ DatantIn Ver. ~.1
c210> 1
e211> 1656
c112> DNA
<213> Tetrahymcna thesmophila
<400> 1
atgcaaaaga tacttetaat tactttcett cttggaatag etetegetca aattactcet s0
ggcgttgacc ctatttcagc taaggttacg cctaaaccta agaattacac ttatggagat 120
ctgagcttac tLgtcactga tcettgcgga gtcecttaca gacottctgt tgggtcagga 180
aaageaccca accatgtcta tcaaattatt ggattctaca ctttgaaeat tttcaattct arc
aacgaaaact ctcgtgctat gtaaagagaa ttgcataaga atgasacaaC cattgaaaag 300
atgegtagat tacaacattc ctaaaatata gtcttegata tttttatcta agaagetqet 360
ttggccactg cagacacaet cgaagacgaa tattatgntt cataaattta taataccaea 420
tattggaaat tgaccgetaa caaatatgtt ggtttactcc gtggtttaga aacttactct 480
caattattca cttaagacga agacactgaa gattggtatt cgaataacat ccctacttct 540
atteaagatt aacctgaeta catctacaga ggtettatga cagatteage eagaeattte 600
teatcagtcg aaactatttt aaaaactatt gattctatgt tattcaacaa gttgaatgtt s60
ctccaetQgc acatcactga tactgaatcc ttcccettcc ctcttaaatc attccccaat 7~0
attactaaat aeggagccca ctctaagaag aaacastaca gcttcgaaga catttaatac 780
attgtagact aagctcecaa caagggtatt taagttattc ctgaagtcga ttetccagga B40
cncgettett catgggctag atctcctcaa ttctctagta ttggtctatt atgtgactaa 900
tataatggat agctagaccc aacaetaaat ttaaettaca ccgctgttaa gggtattaeg 9a0
gaagatatga ataettaatt ctacactgct aagtatgttc aeettggtgg tgatgaagtt lOZO
gaagantaat gctggaataa acgccctgaa atcaaggaat tcatgaatta aaataaeatc 1080
tctacatata ctgatttgta gaatcattac agaaagaact aagttaacat ttggaaatca 1140
aetaatgeca etaageetge Lattttetgg gcagaeccaa atactttgas atatggecet 1200
gatgatatta tccaatggtg gggatctact catgattttt cttcaateaa agatcttcat 1a60
aacaaaataa etttatcttt etatgataat acttatttgg atgttggtga gggaaataga 1320
tatggtggaa gttatggcag catgcataac tgggatgtct taaactcttt eaatcetaga 1380
gttectggaa ttaagggtga aattcttggt ggcgaaacat gcttatggag tgaaatgaat 1440
gatgattcta cttaactcta aagactttqg acaagaaata gtgcatttgc tgaaagacte 1500
tggaacaCtg atgctgctaa caatgaaact tacaaaacta gagCtttagt ta$eagaatg 1560
1
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gtctttatgc naCaccgttt aactgetaga ggaatccetg cttcteetg= aacagttggt 16=0
atttgtgaat aaaaCCCttc CctCtgetaC aattga 1656
<Z10> 2
<211> 549
<212> PRT
<Z13> Tetrahymena thermophila
<400> 2
Mez Gln Lya Ile Leu Leu Ile Thr phe Leu Leu Cly Ile Ala Leu Ala
1 5 10 15
Gln Ile Thr pro Gly Val nsp Pro Ile Ser Ala Lys Val Hst Pro Lys
20 25 30
Pro Lye Asn Tyr Thr Tyr Gly Asp Leu 9er Leu Leu Val Thr Asp Pro
35 40 45
Cys Gly vai Ser Tyr Arg Pzo Ser Val c3ly ser Gly Lye Val pro Aan
so ss so
Hss Val Tyr Gln Ile Ile Gly Phs Ty! Thr Leu Asn Ile Fhe Asn 8er
65 'l0 75 80
Aen Glu Aan 9er Cys Ala stet aln Arg Olu Tyr Lye Aan c3lu Thr Thr
05 90 95
Ile Glu Lys Met Rrg A=g Leu Gla Hie ser aln Avn =1e val phe lisp
100 105 1i0
Ile Phe Ile Gla Asp Ala Ala Leu Ala Thr Ala Asp Thr Lau Glu Asp
w lls lzo las
Olu Tyr Tyr Aep Leu Qln Ile Tyr Asn Thr Thr Tyr Trp IryR Leu Thr
130 135 140
Ala Aen Lys Tyr Val Gly Leu Leu Arg c;ly Leu Glu Ths Tyr Ser vln
145 150 155 160
Leu phe Thr Oln Asp oiu Asp Thr Glu Asp Trp Tyr Leu Asn Asa Ile
iss loo ins
Pro ile Ser Ile Gln Asp Gia Pro Acp Tyr Ile Tyr Arg Gly Leu Met
l80 185 190
ile Asp 8er Ala Arg His Phe Leu Ser val Olu xhr ile Leu Lys Thr
195 100 105
Z
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ile Asp Ser Met Leu Phe Asn Lys Leu Asn Val Leu Hip Trp Hic ile
210 215 220
Thr Aep Thr Glu Ser PLe Pro Phe Pro Leu Lys Ser Phe Pro Asn Ile
225 230 235
240
Thr Lys Tyr Oly Ala Tyr ser Lys Lya Lye Gln Tyr Ser Phe Glu Asp
245 250 255
Ile Gln Tyr Ile Val Asp Gln Ala Leu Asn Ly3 Gly Ile Gln Val Ile
260 265 270
Dro c3lu Val Acp ser pro c3ly Iiic Ala Phe Ser Trp Ala Arg Ser pre
275 280 2$5
aln Phe Ser 8er Ile Gly Leu Leu C~~4 Asp Gln Tyr Aaa Gly Gin Leu
290 295 300
Asp Pro Thr Leu Asa Leu Thr Tyr Thr Ala val Lye Gly ile Mec Glu
305 3I0 31s 320
Asp Met Ann T3ir Gln Phe Tyr Th7C Ala Lys Tyr Val His Phe Gly Gly
325 330 335
Asp Glu Val Glu Glu Gla Gars Trp Asn Lys Arg pro Giu ile Lys alu
340 345 350
Phe Men Asn Gln Acn Aan Ile Ser Thr Tyr Tbr Asp Leu aln Asn Tyr
355 760 36S
Tyr Arg Lya Asn Gln Val Asn Ile Trp Lys Ser ile Asn Ala Thr Lyc
370 375 380
Pro Ala Ile phe Trp Ala Asp Ser Aaa Thr Leu Lya ?yr Gly pro Asp
385 390 395 400
Aep Ile x1e Gln Trp Trp Gly ser Thr ~iiv Acp Phe Ser 9er ile Lyc
4os 4i0 sly
Asp Leu Pro Asn Lys iie i1e Leu Ser Phe Tyr Asp Asn Thx Tyr Leu
420 425 430
Acp vat ely Glu o1y Asn Arg ~ryr Gly ply ser Tyr Gly &er Met Tyr
435 440 445
Asri Trp Asp Val Leu Aan Sir Phe Asn Pro Arg Vil Pro aly Ile Lys
450 455 460
3
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Gly alu Ile Leu Gly Glu Thr Gys Leu Trp Ser Glu Met Asn Asp Asp
a65 470 475 480
ser Thr Glri Dhe Gln Arg Leu Trp Thr Arg Asn Ser Ala Dhe Ala Glu
485 490 493
Arg Leu Trp Aan Thr Aap l~la Ala Asn Asa Glu Thr Tyr Lys Thr Arg
500 505 510
Ala Leu val Ser Arg Met val Phe Met Oln Itia Arg hsu Thr Ala Arg
515 SZO 52s
Oly Ile D=O Ala Ser pro Val Thr Val Gly ilc Cyc ~31u Gln Asn Leu
530 535 540
Ser Leu Gya Tyr Asn
545
<210a 3
<Z11> 1837
<212a DNA
<~13> Tetrahymena thermpphila
<400> 3
cageagtaat aaaaaattct aaatatattg sttgtagcta tgcaaaagat actttt:att 60
actttccttc ttggaatagc tctcgcceaa attactbctg gcgttgaccc tatttcagct 1~0
aaggttatgc etaaacctaa gaattacact tntggagatt tgagcttaat tgtcactgat 180
ccttgcggag tctcttacag accttctgtt gggtcaggaa aagtacccaa ccatgtctat 240
caaattattg gattctacac tttgaatatt ttcaattcta aCgaaaactc ttgtgctatg 300
taaagagaat tgtataagaa tgaaaeaacc attgaaaaga tgcgtagatt acaacattcc 360
taaaatatag tcttcgacat ttttatctaa gacgctgctt tggccaatge agacacactc 420
gaagacgaat attatgattt ataaatttat aataccacat attggaaatt gaetgctaac 480
aaatatgttg gtttactccg tggtttagaa acttactctc aattattcac ttaagacgaa 540
gacactgaag attggtattt gaataacatc cctatttcta ttcaagatta acetgactac 600
atctacagag gtcttatgat agattcaQcc agacatttct tatcagetga aactatttta 660
aaaactattg attctatgtt attcaacaag ttgaatgtzc tceattggca catcactgat 7Z0
actgaatect tccecttcce tcttaaatca tteeetaata ttactaaata tggagcetae X80
tctaagaaga aacaatacag cttcgaagac atttaataca ttgtagacta agctctcaac 840
aagggtattt aagttattcc tgaagtcgat tctccaggac acgctttttc atgggctaga 900
tctccttaat cctctagtat tggtctatta tgtgattaat ataatggata gttagaccca 860
acactaaatt tsacttacac tgctgttaag ggtattatgg asgatatgaa tacttaattc lOSO
tacactgcea agtatgtcca ttstggtggt gatgaagttg aaqaataatg ctQgaataaa 10A0
cgccctgaaa ttaaggaatt catgaattan aatascatct ctacatatac tgatttgtag 1140
aattattaca gaaagaacta agetaacatt tggaaatcaa ttaatgetae taagectgct 1=00
attttctggg cagattcaaa tactttgaaa tatggtcctg atgacattat tcaatggtgg 1260
ggatctaete atgattttce ttcaatcaaa gatcttccta acaaaataat CCtatettte 1320
4
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taegataata cttatttgga cgttggegag ggaaatagat atggtggaag ttatggeagc 1380
atgtataact gggatgtctt aaactctttc aatcctagag ttcctggaat Caagggtgaa 1440
attcttggtg gcgaaaeatg cttatggagt gaaatgaatg atgattctac ttaattctaa 1800
agactttgga caagaaatag tgcatttgct gaaagacttt ggaacactga tgctgceaac 1560
aatgaaactt acaaaactsg agcttcagtt agcagaatgg tctttatgca acaccgttta 1620
actgctagag gaatccctgc ttctcctgta acagttggta tttgtgsata aaacctttct 1680
' ctctgetaca ategattcta aatataaara ttaaataaat attttaagaa aeatttetaa 140
gaatatttta gtataaaaac tgtattttaa ttgataaaaa aaatataaat attattatta 1800
.. attgaacttt agctaaaaaa aaaaaaaaaa aaaaaaa 1837
..
RECEIVED TIh~ RLIG.24. 5-2'Tf~1 PRINT TII'~ HLIG.24. 5:33RM