©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
C-terminal Extension of Truncated Recombinant Proteins in Escherichia coli with a 10Sa RNA Decapeptide(*)

Guo-Fen Tu , Gavin E. Reid , Jian-Guo Zhang , Robert L. Moritz , Richard J. Simpson (§)

From the (1) Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch) and the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)() from E. coli (8) we observed that 5-10% of the mIL-6 molecules contained a novel C-terminal modification.

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.


EXPERIMENTAL PROCEDURES

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 proABlacI, 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) .


RESULTS AND DISCUSSION

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.


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.

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).


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.

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.


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.


FOOTNOTES

*
This work was supported by National Health and Medical Council of Australia Grant 920528 (to R. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia. Tel.: 61-3-347-3155; Fax: 61-3-348-1925; E-mail: simpson@licre.ludwig.edu.au.

The abbreviations used are: mIL-6, murine interleukin-6; hIL-6, human interleukin-6; PCR, polymerase chain reaction; RP-HPLC, reversed-phase high performance liquid chromatography; CNBr, cyanogen bromide; ESI-MS, electrospray ionization-mass spectrometric analysis; cat, chloramphenicol acetyltransferase gene; ssrA, chromosomal 10Sa gene; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; kb, kilobase(s).


ACKNOWLEDGEMENTS

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.


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