From the
When murine interleukin-6 is overexpressed in Escherichia
coli, a small population of molecules exhibits a novel C-terminal
modification. Peptide mapping, electrospray ionization-mass
spectrometry, and automated N- and C-terminal sequencing identified a
peptide (``tag'' peptide),
-Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small
metabolically stable RNA of E. coli (10Sa RNA) attached to
truncated C termini of the recombinant protein. A mutant strain of
E. coli in which the chromosomal 10Sa RNA gene ( ssrA)
is disrupted does not produce this C-terminal modification, confirming
that the tag peptide originates from the ssrA gene.
Recombinant proteins produced in Escherichia coli occasionally contain structural modifications that restrict their
usefulness as therapeutic drugs or reagents for structure-function
relationship studies. Such modifications include N- and C-terminal
truncations
(1, 2) , extensions
(3) , incomplete
removal of N-terminal initiator methionine
(4) , trisulfide
derivatives
(5) , misincorporation of lysine for arginine
(6) , and norleucine for methionine
(7) . During the
purification of recombinant murine interleukin-6 (mIL-6)
We report here the identification of a population of
C-terminally truncated mIL-6 molecules in our recombinant mIL-6
preparation that contain an appended peptide
(-Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH) at their C termini.
Our discovery that this C-terminal ``tag'' peptide is encoded
by a small metabolically stable RNA of E. coli (10Sa RNA)
(9) and that its formation in mIL-6-tag chimeras is not
dependent upon the bacterial strain or plasmid employed has novel
structural and functional implications and reveals a hitherto
undescribed biochemical process.
Bacterially expressed mIL-6
(19) was purified from
inclusion bodies by RP-HPLC and characterized by peptide mapping,
microsequencing, and mass spectrometry
(8) . In addition to the
full-length molecule, two other analogues, designated T1 and T3, with
identical N-terminal sequences to mIL-6 were observed. Although
apparently homogeneous when chromatographed on a Brownlee C8 column,
SDS-polyacrylamide gel analysis showed that T1 and T3 were of lower
apparent mass than the 20-kDa mIL-6 and exhibited mass heterogeneity
(8) . Capillary RP-HPLC-electrospray ionization-mass
spectrometric analysis (ESI-MS) confirmed that T1 and T3 were complex
mixtures, comprising molecules in the mass range 18-20 and
13.5-15 kDa, respectively. Intriguingly, automated C-terminal
amino acid sequence analysis yielded a single sequence,
-Leu-Ala-Ala-COOH, which bore no relationship to mIL-6
(19) (Fig. 1). Thus, we were confronted with a series of
mIL-6 proteins with the same N-terminal and C-terminal sequences but
with a spectrum of molecular masses.
Inspection of the tag peptide sequence showed
that it is unrelated to either mIL-6
(19) or pUC9 sequences
(20) but is identical to the last 10 amino acid residues of a
putative polypeptide encoded by a small metabolically stable RNA (10Sa
RNA) in E. coli (9) . The ssrA gene has its
own promoter and terminator and an open reading frame encoding a
polypeptide of 25 amino acids
(9) . Thus far, attempts to
identify the 10Sa RNA polypeptide in E. coli extracts have
been unsuccessful
(21) . While the function of 10Sa RNA is not
clear, disruption of the ssrA gene has been shown to retard
cell growth
(21) .
To ascertain whether the tag peptide
actually originates from the 10Sa RNA, we disrupted the ssrA gene in E. coli strain JM101 containing the mIL-6 plasmid
and analyzed for the presence of mIL-6-tag peptide chimeras. Using a
donor strain of E. coli (N2211), in which ssrA had
been disrupted by insertion of cat into the 10Sa RNA coding
sequence
(21) , we transferred the disrupted ssrA gene
from the donor chromosome to that of JM101 by P1-mediated transduction
using P1 vir (11) . After transfer, a
chloramphenicol-resistant colony, NM101, was selected for
transformation with the mIL-6-containing plasmid p9HP1B5B12. Two
chloramphenicol- and ampicillin-resistant transformants, NM101-1
and NM101-2, were chosen for further study. PCR analysis of DNA
from single colonies of NM101-1 and -2 verified the insertion of
cat into ssrA (Fig. 2). Using primers specific
for ssrA, a 0.36-kb PCR product corresponding to the expected
fragment length of ssrA was amplified from strain JM101
(Fig. 3, lane 1). For NM101-1 and -2, the
1.1-kb PCR product obtained is in agreement with a segment comprising
the 0.36-kb ssrA and a 0.8-kb fragment from cat (Fig. 3, lanes 2 and 3). The
presence of the mIL-6 plasmid in strains NM101-1 and -2 was
confirmed by PCR amplification of a 0.5-kb product using mIL-6-specific
primers (Fig. 3, lanes 5-7).
To
explore whether the tag is added to mIL-6 during RNA synthesis via a
hitherto unknown co-transcriptional process, we attempted to identify
mIL-6-tag chimeras at the RNA level. A Northern (RNA) blot of total RNA
from E. coli strain JM101 carrying the mIL-6 plasmid
p9HP1B5B12
(19) revealed an abundance of the 0.5-kb 10Sa RNA
(Fig. 5 A, lane 5) but no evidence of
mIL-6-tag chimera transcripts. Disrupted 10Sa RNA transcripts were
undetectable in strain NM101-1 (Fig. 5 A, lane 4). It is evident from the 0.2-0.9-kb smear in
Fig. 5A ( lanes 1 and 2),
however, that a significant portion of mIL-6 mRNA transcripts is
truncated. Using a panel of mIL-6 cDNA oligonucleotide probes, the
truncation was shown to arise from the 3` end of the transcripts
(Fig. 5 B). This may account for the progressive
truncation of mIL-6 from its C terminus (). Reverse
transcription of total RNA followed by PCR, using a sense primer from
the 5` end of mIL-6 cDNA (probe 1) and an antisense primer from the tag
peptide, failed to detect mIL-6-tag peptide chimera RNA transcripts;
this was confirmed by primer extension
(22) using the
tag-peptide oligonucleotide as primer (data not shown). These data
cannot completely exclude the possible existence of a very low level of
chimeric RNA or highly unstable chimeric RNA. Earlier attempts, by
others
(23) , to demonstrate direct binding of 10Sa RNA to
ribosomes proved unsuccessful; hence it appears unlikely that 10Sa RNA
is a mRNA.
We are grateful to J. Pittard and J. Praszkier for
help and advice, C. Miller for performing the C-terminal sequence
analysis and J. Eddes for artwork, and A. W. Burgess and D. S. Dorow
for helpful discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
from E. coli (8) we observed that
5-10% of the mIL-6 molecules contained a novel C-terminal
modification.
Materials
The plasmid p9HP1B5B12 was constructed
by inserting a 0.9-kb mIL-6 cDNA into the EcoRI site of
high-copy vector pUC9
(10) . The pGEX vector carrying
hIL-6 cDNA was a gift from T. Wilson (Walter and Eliza Hall Institute,
Melbourne); the pUC8 vector carrying hIL-6 cDNA was a gift from A.
Hammacher (Ludwig Institute, Melbourne).
Bacterial Strains and Growth Conditions
The
genotypes of the E. coli strains used are as follows: JM101,
sup E thi (lac-proAB) F`[traD36
proAB
lacI, lacZ
M15];
NM522, (hsd
5,
(lac-pro),
thi(F:pro
, lacI, Z
M15, tra
D-35)). N2211 is identical to strain JC 7623 ( recB21, recC22,
sbcB15, sbcC201, thr-1 ara-14 leuB6,
(gpt-proA) 62
lacY1, tsx-33 supE44 galk2,
, rac
,
his G4, rfbD1, rpsL31, kdgK51, xyl-5 mtl-1, argE3, thi-1), except
that the chromosomal 10Sa gene ( ssrA) is disrupted by
insertion of the 0.8-kb chloramphenicol acetyltransferase
( cat) gene into the 10Sa RNA coding sequence
(21) (a
kind gift from B-K. Oh, Washington University, St. Louis, MI). E.
coli strain NM101 containing a disrupted ssrA gene was
derived from strain JM101 by P1 transduction
(11) using N2211
as a donor strain. Bacterial strains were propagated in either
Luria-Bertani (LB) or 2
TY medium at 37 °C with shaking.
Antibiotic concentrations were: ampicillin (40 µg
ml
) and chloramphenicol (40 µg
ml
);
isopropyl-thio-
-
D-galactopyranoside concentration was 0.1
m
M.
DNA Sequence Analysis of ssrA
Reverse
transcription of ssrA was performed using an antisense primer
from ssrA (tag probe) (position 247 to 267;
5`-TGCTAAAGCGTAGTTTTCGTC-3`)
(9) followed by polymerase chain
reaction (PCR) using the same antisense primer and a sense primer from
position 146 to 172 (5`-CACATTGGGGCTGATTCTGGATTCGAC-3`). The amplified
cDNA fragment was cloned into the EcoRI- BamHI site of
the pUC8 vector and sequenced using an Applied Biosystems model 370A
automated DNA sequencer.
Antibody and Immunoblot Analysis
Polyclonal
antiserum was raised against a synthetic tag peptide (AANDENYALA),
synthesized using optimized solid-phase peptide synthesis methodologies
as described
(12) . For immunoblot analysis, proteins were
electroblotted from precast 10-20% Tricine SDS-acrylamide gels
(Novex) onto nitrocellulose paper (Schleicher and Schüll), blocked
with 3% bovine serum albumin and 0.02% Tween 20 in phosphate-buffered
saline for 1 h, and then reacted with the anti-tag peptide polyclonal
antiserum (1:15,000 dilution) followed by incubation with a 1:20,000
dilution of goat antibody to rabbit immunoglobulin G cross-linked
horseradish peroxidase (Amersham Corp.). Immunoblots were developed
using the enhanced chemiluminescence procedure (Amersham,
Buckinghamshire, UK).
Northern Blot Analysis
E. coli RNA was
isolated using the ``hot phenol'' extraction method
(13) . Northern blot analyses were performed as described
elsewhere
(14) . Prehybridization of the blots was performed at
a temperature 5 °C below the estimated melting temperature for the
individual oligonucleotide. Antisense oligonucleotide probes used for
hybridizing to various regions of mIL-6 mRNA
(15) were: probe
1: 5`-GAAACCATCTGGCTAGGT-3`, nucleotides 970-953; probe 2:
5`-GTGTCCCAACATTCATATTGTCAG-3`, nucleotides 798-772; probe 3:
5`-ACTAGGTTTGCCGAGTAGA-3`, nucleotides 670-650; probe 4:
5`-ACCTCTTGGTTGAAGATATG-3`, nucleotides 503-484; probe 5:
5`-ATATCCAGTTTGGTAGCA-3`, nucleotides 345-328; probe 6:
5`-ACAGGTCTGTTGGGAGTGGTATCCTC-3`, nucleotides 167-142. The
antisense oligonucleotide from ssrA (tag probe) was
5`-TGCTAAAGCGTAGTTTTCGTC-3` (nucleotides 267-247)
(9) .
Oligonucleotide probes were end-labeled using T4 polynucleotide kinase
and [-
P]ATP. Hybridization was performed in
the same solution for 17 h at the same temperatures used for
prehybridization. After washing the filters with 6
SSC at 25
°C, bound oligonucleotide probes were visualized by PhosphorImage
analysis using a Molecular Dynamics (Palo Alto, CA) instrument.
Expression and Purification of mIL-6 Analogues T1 and
T3
Recombinant mIL-6 and analogues T1 and T3 were expressed in
E. coli, extracted from inclusion bodies with 6
M guanidinium HCl and purified by gel permeation chromatography as
described
(8) .
Cyanogen Bromide Peptide Mapping of T3, T1, and
mIL-6
Proteins (50 µg) were incubated with a 500-fold excess
of CNBr (w/w of total protein) in 70% aqueous formic acid containing
0.02% Tween 20 under nitrogen for 18 h in the dark at 22 °C.
Capillary RP-HPLC/Electrospray Mass
Spectrometry
Mass spectra were recorded on a Finnigan-MAT
TSQ-700 (San Jose, CA) triple quadrupole mass spectrometer equipped
with an electrospray ion source and a capillary RP-HPLC column (0.2 mm
internal diameter 150 mm) slurry-packed with Brownlee RP-300
(300-Å pore size) 7-µm dimethyloctyl silica (Applied
Biosystems, Foster City, CA)
(16, 17) .
N- and C-terminal Amino Acid Sequence
Analyses
N-terminal amino acid sequence analyses were performed
using Applied Biosystems (models 470A and 477A) instruments. Automated
C-terminal protein sequence analyses of T1 and T3 were performed using
a Hewlett-Packard (model HP G1009A) sequencing instrument
(18) .
Figure 1:
Automated C-terminal
amino acid sequence analysis of mIL-6 analogue T1. Approximately 10
µg (500 pmol) of T1 was taken for sequence analysis, and 80% of the
total thiohydantoin-derivative for each cycle was taken for
thiohydantoin analysis. Thiohydantoin-derivatives are indicated by the
one-letter notation used for amino acids. Representative cycles, along
with the elution profile of a calibration mixture (100 pmol) of
thiohydantoin-derivatives (bottom right-hand corner), are
shown.
To resolve this paradox, Asp-N
endoproteinase peptide mapping was performed, and the peptide maps of
T1 and T3 were compared with that of full-length mIL-6
(8) .
This study showed that T1 and T3 were C-terminally truncated forms of
mIL-6, lacking about 20 and 60 C-terminal residues, respectively.
Further characterization was provided by CNBr peptide mapping (Fig. 2),
which showed that both T1 and T3 lack the parent C-terminal CNBr
peptide, CN0 (Lys-Thr
). Sequence
analysis of the C-terminal peptide from T3 (CN3) confirmed the native
sequence of mIL-6 from residues 101 to 122 and established a novel tag
peptide extension -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH
from residues 123 to 133 (). Heterogeneity at positions 122
(Thr and Ala), 128 (Asn and Tyr), and 130 (Ala and Leu) showed that
peptide fraction CN3 contained two peptides that differed by the
deletion of one residue (Thr
). This was confirmed by
ESI-MS of CN3, which showed two peptides of equal abundance with masses
of 3287 and 3388 daltons (). Edman degradation of the
C-terminal CNBr peptides CN1, CN2, and CN4-8, together with
ESI-MS, confirmed that each contained the sequence of mIL-6 (residue
104 onward) with the tag peptide extension at their C termini
(). In summary, T1 and T3 comprise mIL-6 molecules that
have been progressively truncated in a ``ladder-like'' manner
from the C terminus (in the regions of residues 158-169 and
112-125, respectively) and modified by appendage of a tag
peptide. There was no evidence of any T1 and T3 deletants that did not
contain the tag peptide and no tag peptide shorter than 11 amino acid
residues. The tag peptide has not been found attached to the
full-length mIL-6.
Figure 2:
CNBr
peptide mapping of mIL-6 analogues T1 and T3. The CNBr digest mixtures
(50 µg of protein) of T3, T1, and mIL-6 were evaporated to
near dryness by centrifugal lyophilization, diluted with 0.1% aqueous
trifluoroacetic acid, and applied to a Brownlee RP-300 column (4.6 mm
internal diameter
100 mm), which was developed at 1 ml/min
using a linear 60-min gradient from 0 to 100% B, where solvent A was
0.1% aqueous trifluoroacetic acid and solvent B was 60% acetonitrile,
0.09% aqueous trifluoroacetic acid. Annotated peaks were collected
manually and subjected to automated Edman degradation and ESI-MS.
AU, absorbance unit.
Figure 3:
Disruption of the ssrA gene in
E. coli strain JM101. PCR amplification of the disrupted
ssrA (sense primer, 5`-CACATTGGGGCTGATTCTGGATTCGAC-3`,
nucleotides 146-172; antisense primer,
5`-GAGTTGAACCGCGTCCCGAAATT-3`, nucleotides 499 to 476 (9)) and mIL-6
cDNA (sense primer, 5`-ACCACTTCACAAGTCGGA-3`, nucleotides
172-189; antisense primer, 5`-CACTAGGTTTGCCGAGTAGA-3`,
nucleotides 671 to 652 (15)) was performed from single bacterial
colonies. PCR products were electrophoresed on a 1% agarose gel and
visualized by staining with ethidium bromide. PCR products of the
ssrA gene from strain JM101 ( lane 1), strain
NM101-1 ( lane 2), strain NM101-2
( lane 3) are indicated as well as PCR products of
mIL-6 cDNA from E. coli strain NM101-1 ( lane 5), NM101-2 ( lane 6), and plasmid
p9HP1B5B12 ( lane 7). The fragment lengths (base pairs
( bp)), determined from DNA markers ( lane 4),
are indicated at the left margin.
Having
established that the ssrA gene was disrupted in E. coli strain NM101-1, we overexpressed mIL-6 in this strain as
outlined in Ref. 8 and analyzed the purified mIL-6, T1, and T3 for the
presence of mIL-6-tag peptide chimeras using polyclonal antiserum
raised against the synthetic tag peptide. Western blot analysis shows
that T1 and T3 from strain JM101, but not from strain NM101-1,
contain the tag peptide (Fig. 4 A). ESI-MS analysis of
the C-terminal CNBr peptides of T1 and T3 from NM101-1 confirmed
that they were progressively truncated at the C terminus but did not
contain the tag peptide extension (data not shown).
Figure 4:
Immunoblot analysis of C-terminally
truncated forms of mIL-6 and hIL-6 from various strains of E. coli harboring different plasmids with polyclonal antiserum to the tag
peptide. A, RP-HPLC-purified T1 and T3 (2
µg/ lane) were analyzed by SDS-polyacrylamide gel
electrophoresis using precast 10-20% Tricine gels (Novex).
Lane 1, T3 from NM101-1; lane 2, T1 from NM101-1; lane 3,
full-length mIL-6 from NM101-1; lane 4, T3 from
JM101 containing p9HP1B5B12; lane 5, T1 from JM101
containing p9HP1B5B12. B, immunoblot of insoluble
crude cell lysates (
20 µg of protein/ lane) from
various strains of E. coli harboring different plasmids.
Lane 1, strain JM101 transformed with pGEX
containing hIL-6; lane 2, strain NM101-1
transformed with pUC8 containing hIL-6; lane 3,
strain JM101 transformed with pUC8 containing hIL-6; lane 4, strain NM522 transformed with pUC9 containing mIL-6;
lane 5, strain NM101 transfected with pUC9 containing
mIL-6; lane 6, strain JM101 transformed with pUC9
containing mIL-6; lane 7, fraction T1; lane 8, parent strain JM101. The open arrow indicates the position of full-length interleukin-6 in pUC8 and -9
plasmids; the solid arrow indicates the position of
the 46-kDa full-length glutathione S-transferase-hIL-6 fusion
protein. Molecular mass markers are given to the right in
kilodaltons.
We also
investigated whether protein-tag peptide chimera formation is dependent
upon the recombinant protein, bacterial host strain, or plasmid
employed. Plasmids pUC8, pUC9, and pGEX containing either mIL-6 or
hIL-6 were transformed into E. coli strains JM101, NM101, and
NM522, and the cells were grown as outlined in Ref. 8. Antisera
generated to the synthetic tag peptide identified, by Western analysis,
a number of 18-20-kDa proteins in the insoluble cell pellets from
JM101 and NM522, variously transformed with pUC8, pUC9 containing
either mIL-6 or hIL-6 or, in the case of pGEX, 21-25-kDa proteins
(Fig. 4 B). No tag peptides, however, were present in
either E. coli strain NM101 transformed with the same six
plasmid constructs or the parent E. coli strain JM101.
Figure 5:
Northern blot analysis of mIL-6 mRNA and
10Sa RNA from E. coli strains JM101 and NM101. Total RNA (20
µg/ lane) was electrophoresed through a 1% agarose gel,
transferred to a nylon membrane, and probed with antisense
oligonucleotides to various regions of mIL-6 mRNA. Probe 1, nucleotides
970-953; probe 2, nucleotides 798-772; probe 3, nucleotides
670-652; probe 4, nucleotides 503-484; probe 5, nucleotides
345-328; probe 6, nucleotides 167-142; and 10Sa RNA (tag
probe) (see ``Experimental Procedures''). A, strain
NM101-1 containing mIL-6 plasmid p9HP1B5B12 ( lanes 1 and 4); strain JM101 containing p9HP1B5B12 ( lanes 2 and 5); and parent strain JM101 ( lanes 3 and 6) were probed with mIL-6 oligonucleotide
probe 5 ( lanes 1-3) or the tag probe ( lanes
4-6). B, total RNA (20 µg/ lane) from
strain JM101 containing plasmid p9HP1B5B12 was analyzed on Northern
blots probed with mIL-6 oligonucleotide probes 1-6 ( lanes 1-6, respectively) and tag probe ( lane 7). The positions of 23 and 16 S ribosomal RNA are
indicated.
Alignment of the 25-residue putative amino acid sequence
of the ssrA gene product
(9) and the tag peptide
sequence reveals the presence of an additional alanine residue in the
tag peptide (Fig. 6). To exclude the possibility of a
valine/alanine error in the original sequence, we sequenced the
ssrA from E. coli strain JM101. Our results, which
concur with the published ssrA gene sequence
(9) ,
confirm that no codons have been replaced in the ssrA gene
from E. coli strain JM101. It appears that a Val/Ala
substitution (one base change) may be implicated in the mechanism
underlying the ligation of the tag peptide to the progressively
truncated mIL-6. In this regard, it has been suggested recently that
the RNA encoded by the ssrA gene can fold into a tRNA
shape and can be charged with alanine in vitro (24) .
Figure 6:
Alignment of tag peptide sequence with
predicted sequence from 105a RNA.
Taken together, our studies suggest that formation of recombinant
protein-tag peptide chimeras is dependent on the 10Sa RNA gene. Since
we were unable to detect either mIL-6-tag peptide chimera mRNA
transcripts or the presence in E. coli of the 10Sa RNA peptide
product or precursor protein, the biochemical mechanism underlying the
fusion of the interleukin-6 and the translated 10Sa RNA peptide must
involve a hitherto undescribed biochemical process.
Table: 0p4in
Underlined sequences were obtained by
analysis of Asp-N peptides from mIL-6 fraction T1 (8); peptides CN3/4
were found in both fractions CN3 and CN4; CN5/6 in fractions CN5 and
CN6; and CN4/5 were found in fractions CN4 and CN5. ``Tag''
sequence peptide is designated by boldface italic lettering.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.