gpl20

P.stl

Synthesized ^DNA

1

^PCR

EcoRl EcoRl

�- · · I

T7 RNA Polyhedrin polymerase 5 · UTR promoter

Polyhedrin 5'U1R T7RNA polymerase promoter EcoRl

gpl20

�

17wl

1

^{PCR P"l}

I sssssssss J

Polyhedrin 3'UTR

Polyhed:rin

Pstl

Synthesized ^DNA

1

^PCR

EcoRI _Kpni

�- · · I

T7 RNA Polyhedrin polymerase 5' UTR promoter

gpl20

Polyhedrin

3' ^UTR

5' Uffi

Kpn8,

^Sa!IXhoi Polyhedrin

pgpl20Lls 3'UTR Pstl

EcoRI Pstl

�

^{ln vitro} t ranscription

�

^{[n vitro} t ranscription I.

Polyhedrin 5'UTR

S\SSS\9

gpl20wt Polyhedrin Polyhedrin

3' UTR 5' l.JTR gp 120wt ^mRNA

gpl20

gpl20Lls ^mRNA

ssssss5J Polyhedrin

3' ^UTR

Fig. 2-1. Construction of the vectors for synthesizing gp 120wt mRNA and gp120L\s mRNA for cell-free protein synthesis and in vitro transcription.

chamber was used to disrupt Sf21 cells, and the cells were exposed to compressed nitrogen gas, which prevents disrupted cells from being oxidized. After equilibration, a sudden decompression disrupted the cells in a controlled manner. The insect cell extract was prepared from the disrupted cells mainly by the method described for wheat germ extract

(

Erickson and Blobel, 1983

)

^. Figure 2-2A shows the time course of gp120 synthesis in the insect cell-free system in the presence of the gp120wt mRNA. By Western blot analysis using a monoclonal anti-gp120 antibody gp120 with a molecular mass of 100 kDa was detected as early as 15 min.

The amount of 100-kDa gp120 protein increased with incubation time.

Quantitation by densitometry showed that the 100-kDa gp120 protein was first detected at 15 min, and that its concentration increased linearly for 25 min

(

^{Fig. 2-2C}

)

. The maximal amount of 100-kDa gp120 protein was detected at 40 min, and further incubation caused the degradation of the 100-kDa gp120 protein to low-molecular mass proteins

(

Fig. 2-2A,

indicated by arrowheads

)

, resulting in a slight reduction in the amount of the 100-kDa gp120 protein. In contrast, when the gp120�s mRNA, which encodes gp120 without the natural signal sequence, was translated, a 53-kDa gp120 protein was synthesized, and the amount of gp120 detected increased for 60 min.

Synthesis of gp120 in an insect, wheat germ, and rabbit reticulocyte cell-free system was examined by Western blot analysis

(

^{Fig. 2-3}

)

^{. When}

the gp120�s mRNA was translated, 53-kDa gp120 was synthesized in insect cell-free system, as seen in the wheat germ and rabbit reticulocyte cell-free systems. In contrast, when the gp120wt mRNA was translated in the insect cell-free system, the 100-kDa gp120 protein was mainly detected, and a small amount of the 56-kDa gp120 protein, corresponding to the

polypeptide backbone of gp120, was also observed, whereas the 56-kDa

(A)

...

Incubation time (min)

(B)

Incubation time (min)

0 5 1 0 15 20 30 40 50 60 0 5 1 0 15 20 30 40 50 60

kDa

-107 - 74

...

:t

^{- 49}

- 36

(C)

₁₀₀

'*

-o (1)

(.)

80

:::1 -o 0

a_ ₆₀

0 C\1

T""

a. Cl 40

0

-c :::1

20

0 E

<l:

0

0 10 20 30 40 50 60 70

Time (min)

Fig. 2-2. Time course of gp120 protein synthesis in the insect cell-free system. Reaction mixtures were incubated with gp120wt mRNA

(

_A

)

or gp120�s mRNA

(B).

Synthesized gpl20 proteins were visualized using a monoclonal antibody and

ECL.

The arrow heads indicate synthesized gp120 protein. The arrows indicate degraded gp120.

Product amounts were determined densitometrically

(C).

_Symbols:

e,

gp120wt mRNA;

0,

gp120�s mRNA.

Insect cell extract

Wheat Rabbit germ reticulocyte extract lysate gp120 mRNA - �s wt - �s wt - �s wt

a..,.

kDa -107 - 74

- 49

- 36

Fig. 2-3. Western blot analysis of gp120 synthesized in cell-free systems.

Reactions were carried out with gp120wt mRNA and gp120�s mRNA for 40 min. Synthesized gp 120 proteins in the same volume of reaction were visualized by Western blot analysis. a and b, gp120 translated from

gp120wt mRNA; c, gp120 translated from gp120�s mRNA.

gp120 protein was only synthesized in the wheat germ and rabbit

reticulocyte systems when the gp120wt mRNA was added. These results suggest that the 100-kDa gp120 protein synthesized in the insect cell-free system is glycosylated.

2-3-3 Glycosylation of synthesized gp120

To confirm whether the 100-kDa gp120 protein is glycosylated, gp120 was treated with endo H, which cleaves N-linked high-mannose and hybrid oligosaccharides. As shown in Fig. 2-4, the molecular mass of gp 120 synthesized in the insect cell-free system decreased from 100 kDa to 61 kDa after endo H treatment, proving that the 100-kDa gp120 protein was

glycosylated with N-linked oligosaccharides, and it was designated as g­

gp120.

2-3-4 Protease protection assay

To provide a reliable indication of the integrity of the processed product in terms of the organelle membranes present, a proteinase

protection assay

(

Walter and Blobel, 1983; Rothblatt and Meyer, 1986

)

^was

conducted. Almost all of the g-gp 120 was resistant to treatment with proteinase K

(

^{Fig. 2-5}

)

. This protection was destroyed by solubilization of the microsomes in 0.5% Triton X-100. However, the gp120 derived from gp120�s mRNA was digested with proteinase without Triton X-100.

These results indicate that translated p-gp120s, which have a signal sequence, were translocated into the lumen of microsomes present in this insect cell extract, where they were modified with oligosaccharides.

2-3-5 Binding activity of gp120 to CD4

To confirm the biological activity of gp 120 produced in the insect

41

gp120 mRNA + +

Endo H +

g-gp120 ...

kDa

-107

• - 49

--36

Fig. 2-4. Degl ycosy lation with en do H. Cell-free protein synthesis was carried out for 40 min with gp 120wt mRNA. The reaction mixture was treated with

( +)

or without

(

^-

)

^{en do H,} then subjected to Western blot analysis. g-gpl20 refers to the glycosylated form of gp120. The arrow indicates the deglycosylated form of gp120.

42

gp120 mRNA �s _wt

Proteinase K + + + + + +

Triton X-1 00 + ^- + +

kDa

g-gp120 .... -107

- 7₄ p-gp120 ....

gp120�s ..,..

-49

Fig. 2-5. Protease protection assay of gp 120 synthesized in the insect cell-free system.

cell-free system, the binding ability of the gp120 monomers to CD4 molecules expressed on the surface of QT6 cells was determined using a flow cytometer. The results indicate that g-gp120 synthesized in the cell­

free system could bind to CD4

(

^{Fig. 2-6B}

)

the same as gp 120 expressed in insect cells using recombinant baculovirus

(

^{Fig. 2-6D}

)

. This observation revealed that most of the glycosylated gp120 synthesized in this insect cell-free system is folded into the proper conformation to provide a CD4-binding site.

2-4 DISCUSSION

The author have reported the construction of a cell-free protein synthesis system derived from insect cells that has the ability to translate exogenous mRNA and synthesize biologically active glycosylated proteins.

The Mini-Bomb cell disruption chamber is a device designed to disrupt biological materials by exposing it to compressed nitrogen gas at the desired pressure and then suddenly decompressing it, causing controlled disruption of the biological materials. The nitrogen-disruption method prevents cytosol proteins from being inactivated by oxidation, which sometimes occurs during mechanical shearing operated in air, such as when a Dounce homogenizer is used. To prepare an insect cell extract that retains

translational and posttranslational activities, the Mini-Bomb cell disruption chamber was used to disrupt the cells uniformly and gently.

As shown in this experiment, glycosylated gp120 was synthesized in the insect cell extract derived from disrupted cells prepared with the Mini-Bomb disruption chamber. In contrast, the 56-kDa gp120 protein, corresponds to the polypeptide backbone of gp120, was only synthesized in an insect cell extract derived from a homogenate prepared using a

motor-(A) 80

c (/) ::I 0

()

0���������

+-' (/) c ::I

() 0

1 oo ¹ o 1 1 o2 1 o3 1 o4

Fluorescence intensity

0��������

1 oo 1 o 1 1 o2 1 o3 1 o4

Fluorescence intensity

(B)80

+-' (/) c ::I

() 0

0��������

c (/) ::I 0

()

1 oo 1 o 1 1 o2 1 o3 1 o4

Fluorescence intensity

0���������

1 oo 1 o 1 1 o2 1 o3 1 o4

Fluorescence intensity

Fig. 2-6. Results of flow cytometry analysis of binding ability of gp120 to CD4 expressed on the surface of QT6 cells. QT6-cells expressing CD4 were incubated with gp120 proteins (thick lines) or were not treated (thin lines and gray areas). (Top panels) Reaction mixture containing the gp120 derived from gp120wt mRNA in the insect cell-free system (B) and reaction mixture without template as a native control (A); (Bottom panels) Culture medium containing the gp120 secreted in VLgp120-infected Sf21 cells (D) and the medium of mock-VLgp120-infected Sf21 cells (C).

driven Potter-Elvehjem homogenizer (data not shown). Thus, the Mini­

Bomb cell disruption chamber is a suitable device to disrupt cell membranes for preparation of insect cell extracts that retain not only their translation component but their posttranslational machinery as well.

When the gp120wt mRNA was translated in the insect cell-free system, the initial rate of glycoprotein synthesis corresponded to that of translation of the gp120�s mRNA, however, the reaction was terminated within 40 min (Fig. 2-2C). The translation of the gp120�s mRNA, on the other hand, continued for 60 min (Fig. 2-2B and C). The earlier termination of glycoprotein synthesis may be caused by depletion of substrates for glycosy lation, i.e., lipid-linked oligo saccharides and nucleotide sugars (Kornfeld and Kornfeld, 1985), in this insect cell extract, not by

degradation or inactivation of the posttranslational machinery, suggesting that glycoprotein synthesis may be prolonged by supplementation with substrates for glycosylation. Moreover, degraded gp120 was observed as incubation time increased (Fig. 2-2), and thus inactivation or removal of proteinase in the insect cell extract may increase the glycoprotein

productivity.

The gp 120 of HIV -1 contains between 20 to 26 consensus Asp­

glycosylation sites in all characterized strains of the virus, and N-linked glycans represent almost 50% of the apparent molecular mass of the protein (Allan et al., 1985; Geyer et al., 1988). When gp120 was expressed in insect cells with recombinant baculovirus, it contained high mannose-type N-linked oligosaccharides, partially or extensively processed from

GlcNAc2Man5 to GlcNAc2Man9 and lacked typical 0-linked

oligosaccharides (Yeh et al., 1993). The g-gp120 synthesized in the insect cell-free system had a molecular mass similar to that of gp120 expressed in baculovirus-infected Sf21 cells that reported by Yeh et al. (1993). Endo H

treatment revealed that g-gp120 synthesized in the insect eel-free system had N-linked oligosaccharides. The glycosylation only occured on the polypeptide that have signal sequence at NH2-terminal, indicating that the insect cell-free system can recognize the signal sequence and glycosylate the nasecnet polypeptides.

Furthermore, protease protection assay indicated that nascent gp120 that have signal sequence was localized within the intact microsome, whereas gp 120 that was deleted signal sequence was localized out of the lumen of microsome. This observation confirmes that the insect cell-free system can recognize signal sequence translocate the nasent polypeptide to microsome,and then glycosylate within the microsome.

Interaction of envelope gp 120 of HIV -1 with its primary cellular receptor CD4 initiates viral entry into the host cell (Dalgleish ^{et al.,} 1984;

Klatzmann ^{et al.,} 1984). A discontinuous structure of gp120 sequences composes the CD4-binding site that requires proper conformation for activity (McDougal ^{et al.,} 1986; Kwong ^{et al.,} 1998). The binding of g­

gp120 to CD4 indicated that the insect cell-free system allows folding and maturation of the synthesized g-gp120 protein to a correct functional conformation.

These observations reveal that this insect cell-free system shows dependable translation of exogenous mRNA, with adequate yield, signal recognition, translocation, glycosylation within intact microsomes and correct folding of proteins.

This cell-free system is prepared from a single source and by a single-step extraction, and it contains almost all translational components with intact posttranslational components. It is completely different from previous reconstituted assay systems, ^i.e., the cell-free translation system supplemented with canine microsomes (Blobel and Dobberstein, 1975;

Walter and Blobel, 1983) and the yeast system (Rothblatt and Meyer, 1986), which sometimes lack some accessory factors required for posttranslation.

The posttranslational mechanisms in insect cells are being increasingly well defined (Jarvis and Finn, 1995), facilitating comparison of the results in a cell-free system with those in whole cells. The role of carbohydrates on glycoproteins has usually been analyzed by comparing glycosylated proteins expressed in living cells with nonglycosylated proteins, or by comparing glycoproteins with enzymatically deglycosylated proteins. However, the use of extraction and deglycosylation procedures may lead to conflicting results because the protein may be denatured during preparation. The use of this cell-free system allows the glycosylated protein to be directly

compared with the nonglycosylated protein without removing the cell membrane. The author expect this cell-free system to be a useful tool for synthesizing proteins for functional analysis and to serve as a model system for elucidating the role of carbohydrates on glycoproteins and the

mechanisms of posttranslational modification.

2-5 SUMMARY

The ability of insect cell extract to produce glycoprotein was examined.

When the gp120 mRNA transcribed from the HIV-1 envelope glycoprotein gp120 gene with T7 RNA polymerase was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in the wheat germ and the rabbit reticulocyte systems. By using of gp120�s deleted a signal sequence as a template, the insect cell-free system synthesized polypeptide backbone of gp120, but not glycosylated. Protease protection assay

48

revealed that the glycosylated protein existed in intact microsome, whereas gp120 from gp12011s was outside of the microsome. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endo H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. These indicated that the insect cell-free system

can recognize the signal sequence, translocate the nascent polypepetide and glycosylate in the intact microsome. Furthermore, glycosylated gp120

was bound to human CD4 molecules expressed on the surface of quail cells.

These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.

49

CHAPTER

3

Establishment and characterization of cell-free translation/

glycosylation in insect cell extract

3-1 INTRODUCTION

Many proteins require posttranslational modification to become functional. To synthesize glycoproteins in the wheat germ extract and the rabbit reticulocyte lysate systems, they must be supplemented with

microsomal fractions prepared from dog pancreas

(

Blobel and Dobberstein, 1975; Walter and Blobel,

1983).

However, these reconstituted assay systems which are a mixture of heterogeneous machineries might lack some posttranslation components. Cell-free glycoprotein synthesis systems have also been constructed from yeast

(

Rothblatt and Meyer,

1986), Xenopus

eggs

(

Matthews and Colman,

1991)

and

Trypanosoma brucei (

Duszenko

et

al.,

1999).

The yeast system is homologous but is supplemented with microsomal fraction prepared from yeast, the

Xenopus

eggs system requires supplementation of

S-100

fraction prepared from rabbit

reticulocyte lysate to increase its translation efficiency, and the T.

brucei

system does not allow initiation of protein synthesis.

In a previous experiment

(

Chapter

2),

the author constructed a cell­

free translation/glycosylation system derived from cultured insect cells by using a Mini-Bomb cell disruption chamber, in which the device produced cell preparation

(

"pressate"

)

from a cell suspension by the introduction of compressed nitrogen gas followed by sudden decompression. The insect cell-free system, which was prepared from a single source and with a single extraction, is different from the traditional reconstituted cell-free assay

systems, such as the rabbit reticulocyte lysate system supplemented with canine pancreatic microsomes. The insect cell-free system can be used to synthesize glycoprotein in the lumen of microsomes contained in the extract, and will be a useful tool for synthesizing functional proteins

in vitro,

and as a model system for elucidating the mechanisms of posttranslational

modification, protein sorting and membrane integration.

This chapter describes in detail the optimal conditions for

glycosylation in the novel insect cell-free system. The optimal conditions for the cell-free protein synthesis reaction were determined, and the cell disruption conditions with the Mini-Bomb were re-optimized according to glycosylation. Moreover, comparison of activities of the extract prepared from insect cells disrupted with the Mini-Bomb and with the Potter­

Elvehjem homogenizer revealed that the Mini-Bomb is more efficient for preparation of extract for this cell-free translation/glycosylation system.

Using this system, about 35 !J,g of glycosylated human immunodeficiency virus type 1

(

HIV-1

)

gp120 protein could be synthesized per ml of the reaction mixture.

3-2 MATERIALS AND METHODS

3-2-1 Cell culture conditions

Spodoptera frugiperda (

IPLB-Sf21-AEII

) (

Vaughn

et al.,

1977

)

was maintained as a spinner culture in IPL-41 medium

(

Gibco BRL Life

Sciences, Grand Island,

NY)

supplemented with 10%

(

v/v

)

heat-inactivated fetal bovine serum

(

Gibco BRL Life Sciences, Grand Island,

NY).

Sf21 cells were routinely maintained at 27aC at densities from 0.1 to 1.5 X 106 cells/mi.

3-2-2 Preparation of insect cell extract

Preparation of the insect cell extract for cell-free protein synthesis was described in Chapter 2. Briefly, the Sf21 cells disrupted by the Mini­

Bomb cell disruption chamber (Kontes Glass Company, Vineland, NJ, USA) were centrifuged for 15 min at 14000 rpm at 4oC in the SW40Ti rotor. The pellet was discarded and the supernatant (approximately 2 ml) was collected and chromatographed through a Sephadex G-25 Fine column (16.0 x 1.0 em), previously equilibrated with column buffer (40 mM Hepes-KOH pH 7.95 at 25°C, 100 mM KOAc, 5 mM Mg(OAc)2, and 4 mM

DTT).

Fractions of 0.5 ml were eluted with the same buffer. The two fractions with the highest RNA/protein concentration were pooled and aliquoted, immediately frozen in liquid nitrogen, and stored at -80°C.

3-2-3 RNA preparation

The pUC19 subcloned EcoRI fragment containing the ^env region of HIV-1sF162 (Cheng-Mayer ^et al., 1990) (GenBank accession number

M38428) was used as the source of the HIV-1 gp120 sequence. The EcoRI-Pstl DNA fragment containing the gp120 coding region flanked by the 5' and 3'-untranslated regions of polyhedrin downstream of the T7 RNA polymerase promoter was cloned into the EcoRI-Pstl sites of pUC18, producing pgp120wt. The pgp120wt was linearlized with Pstl and transcribed with T7 RNA polymerase using an mRNA synthesis kit

(Ambion). The transcript was purified by phenol/chloroform extraction and isopropanol precipitation, and fractionated by formaldehyde agarose gel electrophoresis to check the size and the homogeneity. The samples were resuspended in diethy lpyrocarbonate treated water and stored at -80°C.

3-2-4 Cell-free protein synthesis in insect cell extract

Cell-free protein synthesis was performed with addition of gp120wt mRNA (see in Chapter 2) in the presence of biotinylated lysyl-tRNA for 60 min at 27°C as described in Chapter 2. The reaction was terminated by adding sodium dodecyl sulfate (SDS) gel loading buffer and boiled for 5 min.

3-2-5 Analysis of biotinylated proteins synthesized in insect cell extract

The obtained samples were resolved by 10% SDS-polyacrylamide gel electrophoresis, and biotinylated polypeptides were transferred to a PVDF membrane (Hybond-P, Amersham Pharmacia Biotech). Membrane

blocked by 5% skim milk was incubated with streptavidin-conjugated HRP (Amersham Pharmacia Biotech) for 1 h, then incubated with ECL reagents (Amersham Pharmacia Biotech) for 1 min according to supplier's

instructions. The biotinylated products were visualized by exposure to X-ray film (Fujifilm). Quantitation of products was carried out using a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

3-2-6 Deglycosylation

Deglycosylation with endo-�-N-acetylglucosaminidase H (endo H) was performed as described in Chapter 2.

3-2-7 Western blot analysis

Synthesized and standard gp120 (Immuno Diagnostics, Bedford, MA, USA) were detected by Western blot analysis using a monoclonal antibody (International Enzymes, Fallbrook, CA, USA) as the primary antibody and

53

an anti-sheep lgG-HRP conjugate (Zymed Laboratories, San Francisco, CA, USA) as the secondary antibody. The gp120 proteins were visualized using an ECL chemiluminescence system (Amersham Pharmacia Biotech).

The products were quantified using a scanning densitometer (Molecular Dynamics).

3-3 RESULTS

3-3-1 Glycosylated gp120 synthesized in the cell-free translation/glycosylation system

The insect cell-free translation/glycosylation system prepared from insect cells can translate exogenous mRNA and glycosylate nascent

polypeptides via intact microsomes present in the extract. To test the productivity of this system, an HIV-1 gp120wt mRNA encoding the gp120 protein, which has a polypeptide backbone of 56 kDa and has 21 potential N-glycosylation sites (Asn-Xaa-Ser/Thr), was constructed. The translated

products were detected by incorporation of biotin-labelled lysine.

Incorporation of modified lysine residues carrying the bulky biotin moieties did not interfere with the translation or translocation processes (Kurzchalia et al., 1988). Cell-free protein synthesis in the insect cell-free system using gp120wt mRNA as a template generated two specific gp120 species which migrated at 56 kDa and 100 kDa. Endo H treatment revealed that the 100-kDa gp120 was anN-linked glycosylated protein (g­

gp120) and that the 56-kDa product was precursor gp120 (p-gp120) that had not undergone posttranslational modification (Fig. 3-1 ).

g-gp120 ...

* p-gp120 ...

2 3

kDa -107

- 74

- 49

- 36 - 29

Fig. 3-1. Cell-free glycoprotein synthesis and deglycosylation of synthesized

gp120.

The cell-free protein synthesis reaction was performed with

(

lane

2

and

3)

or without

(

lane

1)

gp

120

mRNA in the presence of biotinylated lysyl-tRNA. The reaction mixture was then treated with endo H

(

lane

3).

The asterisk indicates deglycosylated

gp120.

3-3-2 Optimization of glycoprotein synthesis in the cell-free translation/glycosylation system

The cell-free protein synthesis system contains a number of

components that are essential for their high protein synthesis activity. To increase the glycosylation efficiency of the insect cell-free system, the concentrations of cations, nucleotide triphosphates, CK, CP, and spermidine were optimized using of gp120wt mRNA as a template. The translation efficiency was estimated based on the total amount of g-gp120 and p-gp120, and the glycosylation efficiency was estimated by the amount of g-gp120 synthesized from gp120wt mRNA for 60 min at 27°C.

Generally, when cell-free translation systems were prepared from various cells, the Mg2+ and K+ concentrations required for maximal activity varied considerably (Jackson et al., 1983; Erickson and Blobel, 1983).

The response curves of translation at 100 mM KOAc in the insect cell-free system showed a sharp optimum at 1.4 mM Mg(OAc)2• The response curves of glycosylation also showed a sharp optimum at 1.4 mM Mg(OAc)2 (Fig. 3-2A). For both translation and glycosylation, the optimal K+

concentration was found to be 100 mM (Fig. 3-2B) and response curves for K+ paralleled each other.

ATP and GTP hydrolysis are essential for translation (Huncl ^et al., 1985; Rapaport et al., 1987) and posttranslational protein translocation (Sanz and Meyer, 1989; Bernstein et al., 1989). The optimal ATP

concentrations were found to be in the range of 1.5 mM for translation and 1.75 mM for glycosylation (Fig. 3-2C). However, the productivity of this system was markedly affected by the addition of GTP, and the optimal GTP concentration was found to be 0.25 mM for translation and for

glycosylation (Fig. 3-2D). Furthermore, when ATP and GTP concentrations were varied, the rates of stimulation and inhibition of

56

�

₁₀₀

C/) 80

·u; Q)

.r:. 60

c >.

C/) 40

c

"(i)

+-' ₀ 20 0.. .._

�

₁₀₀

C/) 80

·u; Q) .r:. c 60

>.

C/) 40

"(i) _+-' c

20 0

0::

�

₁₀₀

C/) 80

·u; Q) .r:. ^+-' 60

c >.

C/) 40

c

"(i)

0 20 0::

.-..100

�

C/) 80

·u; Q)

£60

c >.

C/) 40

c

"(i)

0 20

0::

1.4 1.6 1.8 2.0 2.2 2.4

Mg(OAc) 2 (mM)

c

0.5 1 1.5 2 2.5

ATP (mM)

E

500 1000 1500 2000

Creatine kinase (f.A.g/ml)

G

0.2 0.4 0.6 0.8 1 .2

Spermidine (mM)

.-.. 100

�

C/) 80

·u; Q) .r:. 60 c >.

C/) 40 c

"(i)

+-' ₀ 20

0:: 0

0 50 100 150 200 250 300 350

KOAc (mM)

�

₁₀₀

D

C/) 80

·u; Q) .r:. c 60

>.

C/) c 40

"(i) _+-'

0 20

0::

0.5 1.0 1.5 2.0 2.5

GTP (mM)

.-..100

� _F

C/) 80

·u; Q) .r:.

+-' c

>.

C/) c

"(i)

+-' 0

a: 0

0 10 20 30 40 50 60 70

Creatine phosphate (mM)

Fig. 3-2. Effect of various components on protein productivity. Following the protein synthesis with gp120wt mRNA as template, synthesized proteins were visualized using HRP­

streptavidin and ECL. The amount of synthesized g-gp120

(e)

and the amount of translated gp120

(0),

which is the sum of g-gp120 and p-gp120, were estimated by densitometry.

glycosylation were similar to those of translation, suggesting that the glycosylation efficiency is largely determined by the translation efficiency in this insect cell-free system.

Both initiation and elongation of protein synthesis may be influenced by changes in energy charge (Rupniak and Quincey, 1975). In particular, the rate of initiation of translation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios (Hucul ^et al., 1985). Most cell-free translation systems used the energy regenerating system composed of CK and CP, and the energy regenerating system is so active that the mono- or diphosphorylated nucleotides are held at low levels (Hucul ^et al., 1985).

The regeneration system stimulated the translation and glycosylation, and the maximal glycosylation and translation were achieved at the CK

concentration of 800 �--tg/ml (Fig. 3-2E). At that CK concentration, the maximal translation and glycosylation were obtained at the CP

concentration of 10 mM (Fig. 3-2F). Higher concentrations of CP dramatically inhibited the translation, but not the glycosylation.

The optimal concentration for spermidine was found to be 0.125 mM for translation and 0.25 mM for glycosylation (Fig. 3-2G). Both

glycosy lation and translation were inhibited at the spermidine

concentrations of over 0.2 mM. Spermidine stimulated both translation and glycosylation to a similar degree, however the glycosylation required higher concentrations of spermidine than translation.

3-3-3 Influence of disruption conditions with Mini-Bomb cell disruption chamber on gly cosylation

The Mini-Bomb yields the pressate that retain many biological activities. In the Mini-bomb, compressed gas from a standard high­

pressure tank is applied to the biological material at the desired pressure.

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