pMelBac A, B, and C

A Baculovirus Transfer Vector for Secretion

of Recombinant Proteins
Catalog no. V1950-20
Revision date: 13 November 2009
Manual part no. 25-0111

MAN0000027

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2
Table of Contents

Important Information........................................................................................................................ 5

Introduction .................................................................................................................. 6
Overview .............................................................................................................................................. 6
pMelBac Vector.................................................................................................................................... 8
pMelBacCAT ...................................................................................................................................... 10

Methods ...................................................................................................................... 11
Cloning into pMelBac ....................................................................................................................... 11
Guidelines for Expression ................................................................................................................ 14
Technical Support.............................................................................................................................. 16
References ........................................................................................................................................... 17

3
4
Important Information

Shipping and pMelBac A, B, and C, and pMelBacCAT are each supplied at a concentration of
Storage 0.5 μg/μl in 40 μl of 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Store at –20°C.

Product The Certificate of Analysis provides detailed quality control and product
Qualification qualification information for each product. Certificates of Analysis are available
on our website. Go to www.invitrogen.com/support and search for the
Certificate of Analysis by product lot number, which is printed on the box.

5
 Introduction
Overview

Introduction The pMelBac A, B, and C vectors are baculovirus transfer vectors designed to
direct expression of recombinant proteins through the secretory pathway to the
extracellular medium. The signal sequence for honeybee melittin, a highly
expressed and efficiently secreted protein, is used to direct secretion of the
recombinant protein. The pMelBac vector is supplied in three different versions
(A, B, and C) to allow correct in-frame fusion with the melittin secretion signal.

Scientific Secretion levels in Sf9 cells have been reported to range from 1 μg/ml to
Background 10 μg/ml of culture medium (Tessier, et al., 1991). In an effort to improve
secretion efficiency, signal sequences from insect-derived secreted proteins were
tested in Sf9 cells using a baculovirus-based expression system. The α-amylase
signal sequence from Drosophila and the honeybee melittin signal sequence were
tested using the plant protein papain as a reporter protein. As a negative control,
secreted expression of native prepropapain was used. Measuring the
accumulation of the papain precursor in the medium (see below) compared
expression levels from these three constructs. Expression of secreted papain
using the melittin signal peptide was about 5 times higher than the secretion of
papain using the native signal, indicating that the honeybee melittin signal
sequence permits high-level secretion of heterologous proteins in insect cells
(Tessier, et al., 1991).

8
Melittin secretion signal

7 Native secretion signal

a-amylase secretion signal

6
[papain precursor] (mg/l)

0
20 30 40 50 60 70 80 90

Hours postinfection

continued on next page

6
Overview, continued

Recombination The pMelBac transfer vectors contain lacZ and ORF1629 sequences homologous
with Bac-N-Blue™ to those found in Bac-N-Blue™ DNA. Upon co-transfection and recombination,
DNA blue, occ- recombinant plaques are formed. Bac-N-Blue™ DNA is linearized to
remove sequences in ORF1629 that are essential for efficient propagation of the
virus. When recombination occurs between pMelBac and Bac-N-Blue™ DNA,
these essential sequences are restored, and viable, recombinant virus is
produced. The diagram below shows where recombination occurs.

Bac-N-Blue™ DNA
P603 P1629
Bsu36 I Bsu36 I

ORF603 3´ lacZ 5´ ORF1629

PETL PPH P1629

5´ lacZ Gene of Interest ORF1629

pMelBac A,B, and C

pMelBac A, B, and C can only be used with Bac-N-Blue™ DNA (Catalog no.
Important K855-01). They cannot be used with Invitrogen's original linear AcMNPV DNA,
BaculoGold™ (PharMingen) or BacPAK6™ (Clontech) linear AcMNPV DNA.

High Five™ Cells High Five™ cells (Catalog no. B855-01) are particularly well suited for expression
of secreted recombinants proteins. This cell line (BTI-TN-5B1-4) was originally
developed at the Boyce Thompson Institute, Ithaca, NY and originated from the
egg cells of the cabbage looper, Trichoplusia ni, the native host of AcMNPV
(Davis, et al., 1992). This cell line has the following characteristics:
• Grows well in monolayer and doubles in < 24 hours for ease of use
• Adaptable to suspension culture and serum-free medium for protein
purification
• Provides 5-10 fold high-level secreted expression than Sf9 cells (Davis, et al.,
1993)
For more information about High Five™ cells or a protocol for adaptation to
suspension culture, call Invitrogen Technical Service (page 11).

Secretion in High Expression of secreted alkaline phosphatase (SEAP) using its native secretion
Five™ Cells signal was evaluated in eight different cell lines including High Five™, Sf9, and
Sf21. On a per cell basis, High Five™ cells produced 20-fold more protein than
Sf21 cells and 23-fold more protein than Sf9. Since High Five™ cells are larger
than Sf9 or Sf21 cells and the assays were performed on adherent cells, the
amount of SEAP was also determined per milliliter of culture medium. In this
case, High Five™ cells produced 5-fold more SEAP than Sf9 cells and 8-fold more
than Sf21 (Davis, et al., 1993). We recommend High Five™ cells for secretion of
recombinant proteins using the honeybee melittin signal sequence.

7
pMelBac Vector

Contents 20 μg each of pMelBac A, B, and C, lyophilized

Control vector pMelBacCAT, lyophilized

Description pMelBac A (4831 bp), pMelBac B (4821 bp), and pMelBac C (4829 bp) are
baculovirus transfer vectors that contain the honeybee melittin secretion signal.
The pMelBac vector is provided in three different versions for simplified cloning
of your gene in-frame with the melittin secretion signal. The vectors contain
sequences homologous to the lacZ gene and ORF1629 sequences in Bac-N-Blue™
linear DNA, allowing production of blue, recombinant plaques. The polyhedrin
promoter drives recombinant expression of your fusion protein.

Features of The important elements of pMelBac are described in the following table. All
pMelBac features have been functionally tested, except where noted.

Feature
Polyhedrin promoter
Honeybee Melittin Signal Peptide
Multiple Cloning Sites in three
reading frames (A, B, and C)
ORF1629 recombination sequences
lacZ recombination sequences
Baculovirus early-to-late (ETL)
promoter
Small size (4.8 kb, half the size of most Permits efficient and easy cloning of
baculovirus transfer vectors)
Ampicillin resistance gene (β-
lactamase)
pUC origin
Benefit
Allows efficient, high-level expression of
your recombinant protein
Permits efficient, high-level secretion of
your recombinant protein
Allows insertion of your gene in-frame
with the melittin signal sequence for
secretion of your protein
Permits integration of your gene into the
Bac-N-Blue™ DNA and restores the
essential ORF1629 for production of
viable, recombinant virus
Permits integration of your gene into the
Bac-N-Blue™ DNA and production of
blue, recombinant plaques for easy
selection
Allows expression of the intact lacZ gene
to produce blue, recombinant plaques in
insect cells
inserts
Selection of vector in E. coli
High-copy number, growth, and
replication in E. coli

continued on next page

8
pMelBac Vector, continued

Map of pMelBac The figure below summarizes the features of the pMelBac vector. The complete
nucleotide sequence for pMelBac A, B and C may be downloaded from our Web
site (www.invitrogen.com) or requested from Technical Service (see page 11 for
details). Details of the multiple cloning site, including the variable region that
determines the reading frame, are shown on pages 6-8.

BamH I

Hind III
EcoR I
Sac I*

BstB I
Xho I

Kpn I
Nco I
Bgl II
Pst I

Sal I
PPH ATG HBM Signal

PETL Rec
om
bi

na
t
en

tio
5´ lacZ Fragm

nS
pMelBac

e q ue nc e
A, B, C
4.8 kb

n
pU
ici

lli
C p
or i Am
* Sac I is not in
Features of pMelBac B: the "C" version
4821 nucleotides of the vector

Polyhedrin promoter (PPH): bases 7-95

Honeybee Melittin (HBM)
signal sequence: bases 96-158
Multiple cloning site: bases 158-213
Recombination sequences
(ORF 1629): bases 424-1224 (C)
Ampicillin resistance gene: bases 1611-2468
pUC origin: bases 2616-3289
5´ lacZ fragment: bases 4519-3407 (C)
lacZ sequences homologous to lacZ sequences
in Bac-N-Blue™ DNA: bases 4295-3407 (C)
Early-to-Late (ETL) promoter: bases 4821-4520 (C)
(C) = Complementary

9
pMelBacCAT

Description pMelBacCAT is a 5608 bp control vector containing the gene for chloramphenicol
acetyl transferase. It was constructed by digesting pMelBac B with Hind III and
dephosphorylating with calf intestinal alkaline phosphatase (CIAP). A Hind III
fragment containing the chloramphenicol acetyl transferase gene was then
ligated into pMelBac B.

Map of Control The figure below summarizes the features of the pMelBacCAT vector. The
Vector complete nucleotide sequence for pMelBacCAT may be downloaded from our
Web site (www.invitrogen.com) or requested from Technical Service (see page
11).

PPH HBM C
P ET
L AT

Re
t

co
en
5´ lacZ Fragm

mb
pMelBacCAT

ination Seq
5.6 kb

u e nc
e
pU
Co
ri llin
Comments for pMelBacCAT:
Ampici
5608 nucleotides

Polyhedrin promoter (PPH): bases 7-95

Honeybee Melittin (HBM)
signal sequence: bases 96-158
CAT ORF: bases 237-893
Recombination sequences
(ORF 1629): bases 2011-1211
Ampicillin resistance gene: bases 2398-3255
pUC origin: bases 3403-4076
5´ lacZ fragment: bases 5306-4194
lacZ sequences homologous to lacZ sequences
in Bac-N-Blue™ DNA: bases 5082-4194
Early-to-Late (ETL) promoter: bases 5608-5307

10
 Methods
Cloning into pMelBac

General Molecular For help with DNA ligations, E. coli transformations, restriction enzyme analysis,
Biology DNA sequencing, and DNA biochemistry, see Molecular Cloning: A Laboratory
Techniques Manual (Sambrook, et al., 1989) or Current Protocols in Molecular Biology (Ausubel,
et al., 1994).

Maintenance of In order to propagate and maintain pMelBac A, B, and C, we recommend that

pMelBac A, B, and you take each vector, resuspend the lyophilized material in 20 μl sterile water to
C make a 1 μg/ μl stock solution. Store at -20°C.
Use this stock solution to transform a recA, endA E. coli strain like TOP10, DH5α,
JM109, or equivalent.

pMelBac A, B, and C vectors are fusion vectors requiring that you clone your
Important gene in-frame with the melittin signal sequence. We provide three versions of
this vector to facilitate correct fusion of your gene to the signal sequence.
Carefully inspect your gene and the multiple cloning site of each vector below to
clone your gene of interest.

Multiple Cloning Below is the multiple cloning site for pMelBac A. Restriction sites are labeled to
Site A indicate the start of the recognition site. The variable region is the boxed region
located after the honeybee melittin secretion signal. The multiple cloning site has
been confirmed by sequencing and functional testing.
Transcriptional Start
+1
Polyhedrin Forward Sequencing Priming Site
1 GATATCATGG AGATAATTAA AATGATAACC ATCTCGCAAA TAAA TAAG TA

Baculovirus Forward PCR Priming Site

51 TTTTACTGTT TTCGTAACAG TTTTGTAATA AAAAAACCTA TAAAT

Honeybee Melittin Secretion Signal

96 ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTA
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val

BamH I Sac I Xho I

138 TAC ATT TCT TAC ATC TAT GCG GAT C GA TGG GGA TCC GAG CTC
Tyr Ile Ser Tyr Ile Tyr Ala Asp Arg Trp Gly Ser Glu Leu
Melittin Cleavage Site

Bgl II Pst I Kpn I Nco I EcoR I BstB I Hind III Sal I

180 GAG ATC TGC AGC TGG TAC CAT GGA ATT CGA AGC TTG GAG TCG
Glu Ile Cys Ser Trp Tyr His Gly Ile Arg Ser Leu Glu Ser
Baculovirus (+15) Reverse Sequencing Priming Site
222 ACT CTG CTG AAG AGG AGG AAA TTC TCC TTG AAG TTT CCC TGG
Thr Leu Leu Lys Arg Arg Lys Phe Ser Leu Lys Phe Pro Trp

264 TGT TCA AAG TAA AGGAGT TTGCACCAGA CGCACCTCTG TTCACTGGTC

Cys Ser Lys ***

312 CGGCGTATTA AAACACGATA CATTGTTATT AGTACATTTA TTAAGCGCTA

Baculovirus Reverse PCR Priming Site

362 GATTCTGTGC GTTGTTGATT TACAGACAAT TGTTGTACGT ATTTTAATAA

continued on next page

11
Cloning into pMelBac, continued

Multiple Cloning Below is the multiple cloning site for pMelBac B. Restriction sites are labeled to
Site B indicate the start of the recognition site. The variable region is the boxed region
located after the honeybee melittin secretion signal. The multiple cloning site has
been confirmed by sequencing and functional testing.
Transcriptional Start
+1
Polyhedrin Forward Sequencing Priming Site
1 GATATCATGG AGATAATTAA AATGATAACC ATCTCGCAAA TAAA TAAG TA

Baculovirus Forward PCR Priming Site

51 TTTTACTGTT TTCGTAACAG TTTTGTAATA AAAAAACCTA TAAAT

Honeybee Melittin Secretion Signal

96 ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTA
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val

BamH I Sac I Xho I Bgl II Pst I

138 TAC ATT TCT TAC ATC TAT GCG GAT C CG AG C TCG AGA TCT GCA
Tyr Ile Ser Tyr Ile Tyr Ala Asp Pro Ser Ser Arg Ser Ala

Melittin Cleavage Site

Kpn I Nco I EcoR I BstB I Hind III Sal I

180 GCT GGT ACC ATG GAA TTC GAA GCT TGG AGT CGA CTC TGC TGA AG
Ala Gly Thr Met Glu Phe Glu Ala Trp Ser Arg Leu Cys ***

Baculovirus (+15) Reverse Sequencing Priming Site

224 AGGAGGAAAT TCTCCTTGAA GTTTCCCTGG TGTTCAAAGT AAAGGAGTTT

284 GCACCAGACG CACCTCTGTT CACTGGTCCG GCGTATTAAA ACACGATACA

Baculovirus Reverse PCR Priming Site

334 TTGTTATTAG TACATTTATT AAGCGCTAGA TTCTGTGCGT TGTTGATTTA

384 CAGACAATTG TTGTACGTAT TTTAATAATT CATTAAATTT

continued on next page

12
Cloning into pMelBac, continued

Multiple Cloning Below is the multiple cloning site for pMelBac C. Restriction sites are labeled to
Site C indicate the start of the recognition site. The variable region is the boxed region
located after the honeybee melittin secretion signal. The multiple cloning site has
been confirmed by sequencing and functional testing. Note that there is no Sac I
site in this version of pMelBac.
Transcriptional Start
+1
Polyhedrin Forward Sequencing Priming Site
1 GATATCATGG AGATAATTAA AATGATAACC ATCTCGCAAA TAAA TAAG TA

Baculovirus Forward PCR Priming Site

51 TTTTACTGTT TTCGTAACAG TTTTGTAATA AAAAAACCTA TAAAT

Honeybee Melittin Secretion Signal

96 ATG AAA TTC TTA GTC AAC GTT GCC CTT GTT TTT ATG GTC GTA
Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val

BamH I Xho I Bgl II

138 TAC ATT TCT TAC ATC TAT GCG GAT C GA TGG ATC CGA C CT CGA
Tyr Ile Ser Tyr Ile Tyr Ala Asp Arg Trp Ile Arg Pro Arg

Melittin Cleavage Site

Pst I Kpn I Nco I EcoR I BstB I Hind III Sal I

180 GAT CTG CAG CTG GTA CCA TGG AAT TCG AAG CTT GGA GTC GAC
Asp Leu Glu Leu Val Pro Trp Asn Ser Lys Leu Gly Val Asp

Baculovirus (+15) Reverse Sequencing Priming Site

222 TCT GCT GAA GAG GAG GAA ATT CTC CTT GAA GTT TCC CTG GTG
Ser Ala Glu Glu Glu Glu Ile Leu Leu Glu Val Ser Leu Val

264 TTC AAA GTA AAG GAG TTT GCA CCA GAC GCA CCT CTG TTC ACT
Phe Lys Val Lys Glu Phe Ala Pro Asp Ala Pro Leu Phe Thr

306 GGT CCG GCG TAT TAA AACACGATAC ATTGTTATTA GTACATTTAT

Gly Pro Ala Tyr ***

Baculovirus Reverse PCR Priming Site

351 TAAGCGCTAG ATTCTGTGCG TTGTTGATTT ACAGACAATT GTTGTACGTA

401 TTTTAATAAT TC

Melittin Cleavage The melittin signal sequence is cleaved upon secretion after Ala21 in the signal
Site sequence.

13
Guidelines for Expression

Introduction The following guidelines and recommendations are provided for your
convenience. If you need more details about the techniques discussed, refer to
Current Protocols in Molecular Biology, Unit 16.9-16.11 (Ausubel, et al., 1994), The
Baculovirus Expression System: A Laboratory Guide (King and Possee, 1992), or
Baculovirus Expression Vectors: A Laboratory Manual (O'Reilly, et al., 1992).

E. coli Transform your ligation mixtures into a competent recA, endA E. coli strain (e. g.
Transformation TOP10, DH5α) and select on LB plates containing 50-100 μg/ml ampicillin.
Select 10-20 clones and analyze for the presence and orientation of your insert.

Transfection into In addition to your construct in pMelBac, we recommend that you include the
Insect Cells control vector pMelBacCAT as a positive control for expression. Plasmid DNA
for transfection into insect cells must be very clean and free from phenol and
sodium chloride. Salt interferes with the positively charged liposomes,
decreasing transfection efficiency. In addition, any traces of phenol are
detrimental to cell growth.
For methods to transfect insect cells, screen for recombinant plaques, PCR
confirmation of recombinant plaques, and expression, refer to the Bac-N-Blue™
Transfection and Expression Guide and the references listed above.

Important Be sure to isolate recombinant virus containing the CAT control protein as a
positive control for expression.

PCR Analysis You may use PCR to analyze putative recombinant plaques in order to isolate a
pure clone. You will need to calculate the expected size of your PCR fragment
based on the primers you are using.

Expression of To evaluate expression and secretion of your recombinant protein, it is very

Recombinant helpful to have recombinant virus containing the CAT control protein cloned in-
Protein frame with the melittin signal sequence. Expression and secretion of the CAT
control protein will rule out any problem with the cells or the MOI. Every
protein has its own requirements for optimal expression and secretion, and you
need to adjust conditions to optimize expression of your particular protein.

We recommend that you:

MEND
RECOM

• Use cells adapted to suspension culture in serum-free medium for expression

AT
ION

of secreted proteins
• Perform a time course of expression to determine the maximum point of
expression
• Have a detection method for your protein

continued on next page

14
Guidelines for Expression, continued

Assay for CAT You may assay for CAT expression by ELISA assay, Western blot analysis,
Protein fluorometric assay, or radioactive assay.

Guidelines for • Conduct a small-scale time course to test both the expression and secretion of
Expression your protein and the CAT protein control
• Infect cells with an MOI of 5 using a high-titer stock of known titer
• Remove 1 ml samples and assay both the supernatant and the cells for the
presence of your protein or CAT
• Assay every 12-24 hours over 5 days to determine the maximum point of
expression and secretion
Once you have determined the optimal time for expression and secretion, you
may scale-up your expression.

15
Technical Support

Web Resources Visit the Invitrogen website at www.invitrogen.com for:

• Technical resources, including manuals, vector maps and sequences,
application notes, SDSs, FAQs, formulations, citations, handbooks, etc.
• Complete technical support contact information
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16
References

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Seidman, J. G. Smith, J. A., and Struhl, K., eds
(1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York, NY.
Davis, T. R., Trotter, K. M., Granados, R. R. and Wood, H. A. (1992) Baculovirus Expression of Alkaline
Phosphatase as a Reporter Gene for Evaluation of Production, Glycosylation, and Secretion.
Bio/Technology 10: 1148-1150.
Davis, T. R., Wickham, T. J., McKenna, K. A., Granados, R. R., Shuler, M. L. and Wood, H. A. (1993)
Comparative Recombinant Protein Production of Eight Insect Cell Lines. In Vitro Cell. Dev. Biol. 29A:
388-390.
King, L. A. and Possee, R. D. (1992) The Baculovirus Expression System: A Laboratory Guide. Vol. Chapman
and Hall. New York, NY.
O'Reilly, D. R., Miller, L. K. and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual.
Vol. W. H. Freeman and Company. New York, N. Y.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Vol. Cold
Spring Harbor Laboratory Press. Plainview, New York.
Tessier, D. C., Thomas, D. Y., Khouri, H. E., Laliberte, F. and Vernet, T. (1991) Enhanced Secretion from
Insect Cells of a Foreign Protein Fused to the Honeybee Melittin Signal Peptide. Gene 98: 177-183.

©2009 Life Technologies Corporation. All rights reserved.

17
Notes:
Corporate Headquarters
5791 Van Allen Way
Carlsbad, CA 92008
T: 1 760 603 7200
F: 1 760 602 6500
E: tech_support@invitrogen.com
User Manual
For country-specific contact information, visit our web site at www.invitrogen.com