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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 38�46 |
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doi:10.1111/j.1745-7270.2008.00376.x |
Recognition of signal peptide
by protein translocation machinery in middle silk gland of silkworm Bombyx
mori
Xiuyang Guo, Yi Zhang, Xue Zhang,
Shengpeng Wang, and Changde Lu*
State Key
Laboratory of Molecular Biochemistry, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China
Received: September
8, 2007�������
Accepted: October
8, 2007
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30470350)
*Corresponding
author: Tel, 86-21-54921234; Fax, 86-21-54921011; E-mail, [email protected]
To investigate
the functions of signal peptide in protein secretion in the middle silk gland
of silkworm Bombyx mori, a series of recombinant Autographa
californica multiple� nucleopolyhedroviruses containing enhanced green
fluorescent� protein (egfp) gene, led by sericin-1 promoter� and
mutated signal peptide coding sequences, were constructed� by region-deletions
or single amino acid residue� deletions. The recombinant Autographa
californica multiple nucleopolyhedroviruses were injected into the hemocoele
of newly ecdysed fifth-instar silkworm larvae. The expression and secretion of
EGFP in the middle silk gland were examined by fluorescence microscopy and
Western� blot analysis. Results showed that even with a large part (up to 14
amino acid residues) of the ser-1 signal peptide� deleted, the expressed EGFP
could still be secreted into the cavity of the silk gland. Western blot
analysis showed that shortening� of the signal peptide from the C-terminal
suppressed� the maturation of pro-EGFP to EGFP. When 8 amino acid residues were
deleted from the C-terminal of the signal peptide (mutant 13 aa), the secretion
of EGFP was incomplete, implicating� the importance of proper coupling� of the
h-region� and c-region. The deletion of amino acid residue(s) in the h-region
did not affect the secretion of EGFP, indicating that the recognition� of
signal peptide by translocation machinery was mainly by a structural domain,
but not by special amino acid residue(s). Furthermore, the deletion of Arg2 or replacement with Asp in the n-region of the signal
peptide did not influence secretion of EGFP, suggesting� that a positive charge
is not crucial.
Keywords������� signal
peptide recognition; sericin-1; silkworm; Bombyx mori; recombinant
AcMNPV
The recognition of signal peptide by cytoplasmic signal peptide recognition particle (SRP) is the first step in protein� secretion [1-3]. The signal peptide-SRP complex is anchored� to the endoplasmic reticulum (ER) membrane, and the signal peptide is subsequently transferred from SRP to the integral membrane glycoprotein, a signal sequence� receptor (SR) located on the ER membrane close to the translocon, the first gate to the secretory pathway [4,5]. After being directed to the translocon, the nascent protein will be translocated through the translocon co-translationally� or post-translationally, most often the former [6-8]. The signal peptide will then be cleaved from the pro-protein by signal peptidase during the co-translational translocation [9] to form mature secretory proteins that are released into ER lumen and the signal peptide will be further cleaved by signal peptide peptidase [10].
The signal peptide has a canonical structure with positively�-charged amino acid residues at the N-terminal (n-region, 1-5 aa), a hydrophobic core in the middle (h-region, 7-15 aa) and a more polar region with non-polar small amino acid residues at positions -1 and -3 at the C-terminal (c-region, 3-7 aa) [11-14]. It has been reported that properties� of residues at the h/c boundary and +1 position of mature protein can influence the translocation and cleavage� of signal peptide [15,16]. The canonical structure� of a signal peptide is conserved throughout evolution. Based on the common structural features, several� prediction software� programs have been developed, such as the SignalP 3.0 server from the Center� for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP-3.0/#submission) [17].
The silk gland of silkworm is a typical exocrine gland. It is a tubular organ consisting of a single layer of huge polyploid cells that can synthesize and secrete 0.2 g protein� (dry weight) in 3-5 d. It is an attractive model for studying� the mechanism of protein translocation and secretion. Sericin� (ser) takes up approximately 30% of the silkworm cocoon. It mainly consists of six kinds of protein molecules, expressed specifically in the middle part of the silk gland by two genes, ser1 and ser2, through alternative splicing [18,19]. The ser1 gene codes for four major constituents� of sericin. It was thought that the first 19 aa constituted the ser-1 signal peptide [20]. The prediction of ser-1 by SignalP 3.0 indicated that the most probable cleavage site is between positions 21 and 22, with a probability� of 0.591, and another less probable cleavage site is between positions 19 and 20. The function of different� regions of ser-1 signal peptide on the secretion of ser-1 remains unclear. Understanding the recognition� of a signal peptide by protein translocation machinery will facilitate the design of a signal peptide for the secretory expression of foreign genes in the silk gland of Bombyx mori.
In our recent works, we found that some strains of silkworm� are permissive to recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV) [21]. Using rAcMNPV vector, silk gland-specific secretory expression of the enhanced green fluorescent protein� (EGFP) gene in silkworm was achieved [22]. Using EGFP as reporter, the secretion of EGFP can be easily observed with fluorescence microscope. The secretion of fibroin heavy chain of silkworm and the cleavage site of the signal� peptide have been studied [23,24].
In this study, we report the role of different regions of ser-1 signal peptide as recognized by the protein trans�location�� machinery through region-deletions or single amino acid residue deletions in the ser-1 signal peptide.
Materials and
Methods
Mutagenesis of ser-1 signal
peptide
The pSerPEGFP plasmid was constructed previously in our laboratory [22], composed of ser-1 promoter, the coding� sequence for the first 21 aa residues of ser-1, the restricted enzyme sites linker (CTGCAGGCATGC, coding� Leu, Gln, Ala, and Cys), the egfp gene sequence (from ATG to TAA), and the 3'-terminal of ser-1. To construct� the plasmids with signal peptide deleted from its C-terminal, a fragment from a single restricted endonuclease� site in pSerPEGFP (ie, ClaI or SacI) to the mutated signal peptide� coding sequence was amplified by polymerase chain reaction� (PCR) and used to replace the corresponding� normal� fragment in pSerPEGFP. The primers� and amino acid sequences for the mutated peptides� are listed in Table 1. The mutants deleted from the C-terminal of ser-1 signal� peptide included the first 21, 20, 19, 18, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, and 1 amino acid residues of ser-1. Two mutants with the first 21 aa and only the first 1 aa of the ser-1 signal peptide� were used as positive and negative controls, respectively.
As shown in Fig. 1, fusion PCR strategy was applied to construct plasmids with signal peptide mutated by region�-deletions and single amino acid residue deletions within the h-region of signal peptide sequence. The 3'-half of P2 contained� the upstream antisense sequence of the deleted region (or residue), and the 5'-half of P2 contained� the downstream antisense sequence of the deleted� region (or residue). The 3'-half of P3 contained the downstream sense sequence of the deleted region (or residue), and the 5'-half of P3 contained the upstream sense sequence of the deleted region (or residue). The 5' sequence of P3 complemented the 5' sequence of P2. Two fragments that amplified with primer pairs P1/P2 and P3/P4, respectively, were mixed together. After denaturing� and annealing, the mixtures were used as templates, then amplified with P1 and P4 to produce� the fusion fragment. This fragment was used to replace the corresponding fragment in pSerPEGFP through two restricted� sites.
The primers used in fusion PCR are listed in Table 2. Mutants constructed by fusion PCR were named as follows: 9- means the 9th aa was deleted; 9-15 means amino acid residues from 9th to 15th of the original signal peptide were deleted. The amino acid sequences of the mutated signal peptides are also listed in Table 2. These mutants were 9-, 10-, 3-5, 6-8, 6-11, and 9-15. But when the Arg2 was deleted, the mutant was named r-, and when this Arg was replaced with Asp, the resulting mutant� was named r/d.
All PCR products were verified with DNA sequencing, and all cloning processes were identified with restriction analysis.
Construction of rAcMNPVs with
ser-1 signal peptide mutants
We constructed a series of rAcMNPVs containing egfp led by sericin-1 promoter and coding sequences for signal� peptide mutants using the Bac-to-Bac system (Invitrogen, Carlsbad, USA), as described previously [22]. Plasmid pFFa2, modified from pFastBacHTa (Invitrogen) [21], was used to construct the donor plasmid. The ser-1 promoter�-controlled egfp expression cassette with signal peptide mutant was cut by EcoRI and BglII from pSerPEGFP derivatives and ligated into pFFa2 digested by EcoRI and BamHI. The resulting donor plasmids were transformed into Escherichia coli DH10BacDEGT [21] competent cells to produce recombinant bacmids. The bacmids were identified� by PCR as previously described [22]. Verified rAcMNPV bacmids were used for sf-9 cell transfection. Generation and large-scale production of the recombinant baculovirus was achieved according to the instructions of the Bac-to-Bac baculovirus expression systems� manual (Invitrogen) using the sf-9 cell line. Viruses� released into the culture medium from infected cells were collected by ultra-centrifugation at 35,000 g for 60 min. The viruses were resuspended in phosphate-buffered saline� (pH 7.5) supplemented with 1% (V/V) fetal� bovine serum (Gibco BRL, Gaithersburg, USA) and stored at -70 �C [22]. Titers� were determined by a Tissue Culture� Infectious� Dose 50 method as described previously� [21]. The sf-9 cells were maintained in Grace's medium (Gibco BRL) supplemented with 10% fetal bovine serum at 27 �C.
Silkworm larvae inoculation
and fluorescence observation� of silk gland
B. mori larvae (bivoltine race, 54A) were reared on mulberry� leaves at 25 �C. The recombinant baculovirus was injected into the hemocoele of newly ecdysed fifth-instar silkworm larvae with a syringe at 106 pfu/larva. At approximately 5 d post-injection, the green fluorescence in the middle silk gland of the silkworm produced by EGFP were observed and photographed with a fluorescence microscope� (Leica MZ FL III; Leica, Switzerland) after dissection.
In the presence of intact ser-1 signal peptide (21 aa), secreted EGFP was located within the silk gland cavity, and green fluorescence could be seen inside the silk gland; when cutting the silk gland or making an opening in the wall of the middle silk gland dipped in water, secreted green fluorescent protein gradually flowed out to the water, along with silk proteins [Fig. 2(A,B)]. In the absence of signal peptide, the EGFP was located in the single layer of huge cells and no green fluorescence could been seen in the outflow [Fig. 2(C,D)].
Crude EGFP extraction, sodium
dodecyl sulfate-polyacrylamide� gel electrophoresis (SDS-PAGE) and Western blot
analysis
As described previously [22], the silk glands dissected from silkworm
larvae were rinsed with cold double-distilled� H2O several times to get rid of adhesive plasma and cells. The middle
part of the silk glands were then cut off and put into ice-cold double-distilled
H2O.
The secreted green fluorescent� protein in the silk gland cavity gradually
flowed out into the water, along with silk proteins. The silk gland wall was
then separated carefully and the mixtures� of EGFP and silk protein were pound
in water. The soluble part, mainly sericin and EGFP, was collected. The
insoluble part, mainly fibroins, was discarded. The soluble part was then
treated with several cycles of freezing, thawing, and concentration [freezing
at -20 �C, thawing under room temperature; the insoluble sericin was separated
immediately� by centrifugation, and the supernatant� was then concentrated by
lyophilization (SpeedVac Savant, Farmingdale, USA)]. The crude EGFP extracts
from several� silkworms were finally concentrated to the appropriate volume,
then subjected to 15% SDS-PAGE as described by Laemmli [25], and transferred
onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford,
USA) as described previously [26]. Western blot analysis was carried out using
the monoclonal anti-GFP antibody GFP (B-2), sc-9996 (Santa Cruz Biotechnology,
Santa Cruz, USA) and horseradish peroxidase�-labeled sheep anti-mouse secondary
antibody, A-6782 (Sigma, St. Louis, USA).
Results
Recognition of signal peptide by
protein translocation machinery in middle silk gland of B. mori
The ser-1 signal peptide predicted by SignalP 3.0-hidden Markov models (HMM) is the first 21 or 19 aa (Fig. 3). Analysis indicates that the n-region is the first two amino acid residues "mr"; the h-region is aa 3-14, "lvlcctlialaa"; and the c-region is aa 15-21, "lsvkafg" or aa 15-19, "lsvka".
A previous study has shown that amino acid mutation of position -1 might affect the secretion of protein [27]. To validate the importance of the c-region of the signal peptide, we constructed rAcMNPVs to express EGFP that was led by ser-1 signal peptides mutated at the c-region. These mutants encode the first 21, 20, 19, 18, and 15 aa of ser-1, and the ability of these mutants in directing secretion� of EGFP was observed. To our surprise, all of these signal peptide mutants, even the one with 15 aa, directed secretion of EGFP reporter normally as judged by fluorescence observation on silk gland (Fig. 4).
Then further deletions stepwise from the C-terminal of the h-region, or region-deletions and single residue deletions� within the h-region of signal peptide, were carried out. It was found that, when deleting from the C-terminal of the h-region, EGFP could secrete into the silk gland cavity, even if only the first 7 aa of the ser-1 signal peptide remained, whereas EGFP was not secreted with mutants 6 aa* and 5 aa*. These results showed that a large part of the signal peptide of sericin-1 could be deleted with its function in directing secretion remaining intact, although a hydrophobic region is indispensable.
In studying the role of amino acid residues in the hydrophobic� region of ser-1 signal peptide, that is, 3-15 aa "fvlcctlialaal", region-deletions and single residue deletions� showed that almost all signal peptide mutants could direct secretion normally, as seen from the green fluorescent protein distribution profile in the middle silk gland, including mutant 9-15 (Fig. 4), in which the hydrophobic� region was largely deleted. These results indicated� that the SRP recognizes the signal peptide mainly by a structural domain, but not by special amino acid.
Positively-charged amino acid
residue at n-region of signal peptide is not crucial
The canonical structure of a signal peptide has a positively�-charged amino acid residue at the n-region. The importance� of a positively-charged amino acid at the n-region of ser-1 signal peptide was studied in this work. The Arg2 was deleted or replaced by a negatively-charged amino acid residue Asp in mutants r and r/d, respectively. When the rAcMNPVs containing the relative EGFP expression cassettes were injected into the hemocoele of silkworm larvae, the EGFP could be secreted into the silk gland cavity (Fig. 4). It showed that the deletion of Arg or its replacement with Asp in the n-region of ser-1 signal peptide� also did not affect the secretion. These results are consistent� with those of Nothwehr et al [27].
Shortening of signal peptide
influences its cleavage by signal peptidase
Secreted fluorescent protein of different mutants was extracted� and detected by Western blot analysis. The secreted� EGFP in the silk gland cavity was released into water at the first step of preparation. Equal amounts of the crude extracts of fluorescent protein sample from different� mutants were run on 15% SDS-PAGE, then transferred� onto a polyvinylidene difluoride membrane and detected with EGFP primary antibody and a horseradish peroxidase-linked secondary antibody. The Western blot profile of the secreted EGFP, directed by ser-1 signal peptide mutants shortening from the C-terminal, is shown in Fig. 5. When the first 20 or 21 aa remained, EGFP was secreted with the signal peptide cleaved, as judged by the 27 kDa single band of the Western blot profile. When the signal peptide was shortened to the first 19 aa, a very weak band appeared with larger molecular weight, supposed� to be the signal peptide uncleaved pro-EGFP. The amount of pro-EGFP gradually increased along with the shortening of the signal peptide from its C-terminal, and turned into the major band when only the first 9 aa remained, whereas the mature protein, m-EGFP, decreased� gradually and turned into a trace band. These results indicated that coupling of the cleavage of the signal� peptide with the translocation process under physiological� conditions could also be broken. This phenomenon was also found when the signal peptidase activity was interfered� with [17]. Along with the shortening of the signal peptide from its C-terminal, it might be possible that pro-EGFP left the ER membrane before the cleavage of signal peptide, then was released to the ER lumen.
Incomplete secretion of EGFP
reporter led by signal peptide mutant 13 aa
In this investigation, we noticed an incomplete secretion of EGFP led by mutant 13 aa. Part of EGFP was retained somehow and aggregated as irregular spots in the middle silk gland cells (Fig. 6). Those signal peptide mutants with just one amino acid residue difference from 13 aa, that is, 13 aa*, 14 aa, and 14 aa*, could direct secretion of EGFP normally, as seen from the distribution of fluorescent protein� in the middle silk gland. The analysis of signal peptide mutants by SignalP 3.0 showed that the most probable� cleavage site in mutant 13 aa was different from any of the other three mutants, and its probability was only 0.385, whereas it was 0.675 for 14 aa, 0.761 for 14 aa*, and 0.859 for 13 aa* (Table 3). It was proposed that the lower cleavage rate caused the accumulation of EGFP in the cells. This result implied that the proper coupling of different regions of the signal peptide is important for secretion.
Discussion
The c-region of signal peptide has the least length variability and consists of relatively small and neutral polar residues. This region is very important for recognition and cleavage by signal peptidase [29]. The "-3, -1" rule states that residues in positions -3 and -1 relative to the cleavage site must be small and uncharged, and large, bulky residues may reside in position -2. We analyzed functional regions for all the mutants by SignalP 3.0, and the results are shown in Table 3. There are alternative sequences that can be recognized and cleaved by signal peptidase in those mutants. These sites locate either within the ser-1 signal peptide mutants, or in the restriction linker "lqac", or in the EGFP coding sequence. So, deletion at the c-region from 21 to 15 aa did not destroy the secretion� of EGFP. When the original c-region and even a large part� of the h-region of ser-1 signal peptide were deleted, a new c-region� and h-region could functionally fill in. The three amino acid residues, taken as aa -3 to -1, were "vka" or "afg" in ser-1, "lqa" (or "aqa") in linker, and "skg" in EGFP. These all suit the "-3, -1" rule.
Three functional regions of ser-1 signal peptide were investigated using the rAcMNPV-EGFP system. All results� from this work revealed that a large part of the hydrophobic� region could be deleted, the N-terminal positively-charged amino acid residue could be turned into a negative one, and 3 aa from -1 to -3 positions could be altered to other suitable amino acid residue. This means that the recognition� of the signal peptide by SRP and the whole subsequent translocation and secretion� process is highly flexible.
Investigations on the impact of systematic mutation of a signal peptide on its interaction with the protein trans�location� apparatuses are no doubt critical for understanding� their interaction mechanism. Mutation research on eukaryotic� signal peptides for clarification of the importance� of the properties of amino acid residue at certain� positions have largely been done with an in vitro transcription�-translation system together with an extracted canine pancreas microsome� system [30]. Our results showed that the effect� of subtle changes on the signal peptide on its interaction with translocation apparatuses could be studied in vivo, in the middle silk gland of silkworm. The exocrine gland is made up of a single layer of huge polyploid cells with a tubular shape, it is expedient� in secretory condition judgment� by using EGFP reporter and it facilitates the preparation and further analysis of secreted protein.
We have constructed a mutant in which the ser-1 signal� peptide was replaced by the signal peptide of BmcecB, an antibacterial peptide of silkworm that expresses in fat body and is secreted into the hemocoele [31]. The secretion of EGFP directed by BmcecB signal peptide was as normal as that of the ser-1 signal peptide (data not shown). The result indicated that the protein translocation machinery of the middle silk gland can recognize BmcecB signal peptide, and it shares common characteristics with that of other tissues. Thus, this system can also be used as a general system for protein translocation research.
Acknowledgement
We thank Dr. Yuan ZHAO from the
Sericultural Research Institute, Chinese Academy of Agricultural Sciences
(Zhenjiang, China) for kindly providing silkworm eggs and silkworms in this
work.
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