Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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9.2 Production of Protein-based Polymers 475

TABLE

9.2.

Continued

31.

[(GVGIP)io-GVGIPGNGIP]n(GVGVP): n = 30^ 40^

32.

(GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)n(GVGVP) YGSEFELRRQACGRTRAPP-PPPLPSGC:

n =

44% 36^

33.

(GVGVP GVGVP GKGVP GVGVP GVGVP GVGVP)n(GVGVP): n =

55% 36%

22^

34.

[(FEGFPAEGFP)5]n(GVGVP): n = 19^

35.

[(GVGVP)io-GVGRGYSLGIP-(GVGVP)io)]n: n =

12%

36.

[(GVGVP)io-GVGRGYSLGIP-(GVGIP)io)]n: n = 7'

37.

[(GVGIP)io-FPGVGQKRPSKRSKYLIP-(GVGIP)io)]n: n =

11%

38.

[(GVGVP)io-FPGVGQKRPSKRSKYLIP-(GVGVP)io]n(GVGVP): n =

39.

[(GVGVP)io-FPGVGQKRPSKRSKYLIP-(GVGIP)io]n(GVGVP): n =

9% 7%

40.

GKGKAPGK-(GVGVP GVGVP GEGVP GVGVPGVGVP GVGVP)n(GVGVP): n = 1

41.

[(FEGVP)io]n: n =

28%

15% 11,4

42.

[(GVGVP)io-GVGVPGRGDSP-(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2]n(GVGVP): n =

43.

GKGKAPGK-[(GVGVP)io-GVGVPGRGDSP-(GVGVP)io]n: n = 7^

44.

(FEGFP AFGFP)5: n = 33, 29, 26, 25, 23,19,18,16,14,11, 6

45.

[(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2-GVGVPGRGDSP-(GVGVP)io(GVGVAP)8]n: n=

11%

4% 3^

46.

[(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2-(GVGVP)io(GVGVAP)8]n: n =

12% 8%

47.

[(GVGIP)ii-GYGIP-(GVGIP)io]2(GVGIP):Re*: n =

11%

10, 9, 8, 7,

48.

[(FVGEP FVGFP)5]n: n = 18 ,17,12, 2,1

49.

(GIGVP GAGVP GIGVP GIGVP GAGVP GIGVP)n: n = 22% IV

50.

[(GVGIP)ll-GYGIP]n(GVGIP)^:

n =

18%

17,16,15,14,13,12,11%

51.

[(GVGVP)io]n(GVGVP)^:

n =

28,

26,18,17% 16 ,15,14,13%

12,11,

52.

[(GVGIP)io]n(GVGVP)^

n =

32%

25% 21% 15%

9% 6%

53.

{[(GVGIP GEGIP GVGIP)2]2)n: n = 20% 9'

54.

([GVGIP GEGIP (GVGIP)4]2)n: n =

23%

10^

55.

[(GVGIP GEGIP GVGIP GEGIP GVGIP GVGIP)2]n: n = 26% 17% 15^

56.

([(GVGIP GKGIP GVGIP)2]2)n: n = 19,18,17,12

57.

([GVGIP GKGIP (GVGIP)4]2}n: n =

21%

12^

58.

[(GVGIP GKGIP GVGIP GKGIP GVGIP GVGIP)2]n: n = 73,

41,

21,17,14,11

59.

[(GVGIP)8(GYGIP]n: n =

32%

24% 18^

60.

([(GVGIP)5GYGIP]2ln: n = 20% 12% ir

61.

([(GVGIP)2GYGIP]3ln: n = 26,15% 14

62.

[(GVGIP),o]ni-Fn3-[(GVGIP)io]„2: nl = 16 and n2 = 6

63.

(GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)n(GVGVP): n = 42%

23% 22%

18% 16^

64.

AKKKKKKG-[(GVGIP)io]n-VCCC: n = 15^

65.

AKKKKKKG-[(GVGIP)io]n-VCCC: n = 18^

66.

GKGKAPGK-[(GGAP)i2]n(GVGVP): n = 1

67.

[(GGAP)i2]n: n = 1

68.

[(FVGVP FEGVP)5]n: n = 1

69.

([(GVGVP)2-GFGVP]3)n: n = 1

70.

[(GVGIP)ii-GSGIP-(GVGIP)io]n(GVGIP)'': n = 1

71.

[(GVGIP)n-GTGIP-(GVGIP)io]n(GVGIP)': n = 1

72.

[(FFGEP)io]n: n = 1

73.

[(GVGIP)n-GSGIP]n(GVGIP)*': n = 1

74.

[(GVGIP)n-GTGIP]n(GVGIP)'': n = 1

75.

[(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2-GVGVPGRGDSP-(GVGVP GVGVP GKGVP GVGVP

GVGFP GFGFP)2]n: n = 1

76.

(GIGVP GAGVP GKGVP GIGVP GAGVP GIGVP)n: n = 1

77.

(GIGVP GAGFP GKGFP GIGVP GAGVP GIGVP)n: n = 1

78.

(GIGVP GAGFP GKGFP GIGVP GAGFP GVGFP)^: n = 1

79.

[ACPGCGGVGIPCPGCG-[(GVGIP)26-Fn3(RGYSLG)-(GVGIP)6]-CPGCGGVGIPCPGCG]„: n = r

^ The protein-based polymer was expressed.

^ The gene was made explicitly using the same codon for a repeating residue in the sequence, and then a different codon

was used in the next repeat.

^ Fn3(RGYSLG) stands for the tenth type III domain of fibronectin in which the cell attachment sequence, GRGDSP,

was replaced by the kinase site, RGYSLG, as a site for phosphorylation.

476 9. Advanced Materials for the Future

looking for a market with future growth. Yeast

has the advantages of excreting the protein-

based polymer from the cell, such that harvest-

ing could proceed without cell destruction, and

a protein-based polymer product from yeast

would not be expected to have such purification

demands as occur with E. coli. Production in the

seeds of a plant such as canola has the advan-

tage of being a value added product where the

base product is the healthful canola oil. In this

case the protein-based polymer would reside

in the protein byproduct that is often sold as

feed for livestock at costs of less than a dollar

per pound. Accordingly, purification from the

protein byproduct could be expected to yield

protein-based polymer at a cost of little more

than that of purification. Because the purifica-

tion process uses a water-based phase separa-

tion, even the remainder of the protein

byproduct could retain its value.

9.2.1.5.2 Elements in the Comparison of

Low-cost Production

Chapter 4 emphasized that biological produc-

tion of protein is energetically an extravagant

process. This represents the basic cost of

biology's capacity to place any of 20 different

amino acid residues at each position and to do

so while ensuring the correct optical isomer.

Such diverse sequences, while maintaining the

same optical isomer at each position, consti-

tutes an impossible feat for chemical synthesis,

and the cost of chemical synthesis yet remains

greater by orders of magnitude.

Despite the very low efficiency for conver-

sion of the energy of photons into the energy

represented by protein that was discussed in

Chapter 4, plants provide the most promising

approach to low-cost production of protein-

based polymers. The cost of harvesting photons

from the sun resides within the cost of main-

taining a healthy plant. Of course, when we

speak of seeking low-cost production of

protein-based polymers, the cost-comparison is

between chemical synthesis and biological syn-

thesis.

Even though chemical synthesis loses

badly in this comparison, specific contributions

of chemical synthesis become invaluable, as dis-

cussed below.

9.2.2 Chemical Synthesis

9,2.2.1 Important Contributions of

Chemically Synthesized Polymers

9.2.2.1.1 Chemical Synthesis Achieved Many

Different Protein-based Polymers Quickly

The approximately 100 polymers that provided

the basis for the data in Figure 5.9 and Table 5.1

were chemically synthesized in about 2 years'

time.

Over the last 10 years, some 80 basic

monomer genes were prepared at BRL. In

general, these monomers were then concate-

merized to produce a multimer gene of the

desired

size.

For proper comparison of the poly-

mers in Figure 5.9 and Tables 5.1, 5.2, and 5.3,

the polymers needed to be of a comparable size.

Therefore, it would have taken 10 years instead

of 2 years to move the basic science to the

understandings provided by the data on chem-

ically synthesized polymers. As shown in Figure

5.10, the data from the chemically sythesized

poly[0.8(GVGVP),0.2(GXGVP)], where X is

either the uncharged Lys (K°) or the uncharged

Glu (E°), are quite comparable with those from

biosynthesized Model Proteins i (E°/OF) and x'

(K°/OF) of Table 5.5. Nonetheless, to obtain the

most quantitative understanding of the com-

prehensive hydrophobic effect as represented

in Figure 5.10, the work should be redone with

biosynthesized protein-based polymers. Suc-

cinctly

stated,

chemical synthesis has moved the

most significant utilization of protein-based

polymers forward by about one decade.

9.2.2.1.2 Comparison of Protein-based

Polymers Having a Random Incorporation of

a Set of Repeats with Those Having the

Repeats in a Fixed Sequence

The profound importance of sequence control

and its relevance to the mechanistic basis of

function could not have been demonstrable

without the capacity to compare the pKa

shifts of Glu residues with a fixed sequence

with those of a random mixture of the same

sequences. For example, the pKa of the chemi-

cally synthesized fixed sequence poly(GVGVP

GVGFP GEGFP GVGVP GVGFP GFGFP)

of 8.1 can be compared with the pKa of

5.2 obtained for the chemically synthesized

9.2 Production of Protein-based Polymers

477

polymer having a random mix of the same

composition of pentamers, namely, poly

[(GEGYF),2(GyGFF),(GFGFF)y' Therefore,

sequence has fundamental consequence. No

wonder biology pays such a high price for the

control of sequence, as detailed in Chapter 4.

9.2.2.1.3 Chemical Synthesis Provided Proof

of Biocompatibility as the Product Had No

Microbial Contaminants

Only because the remarkable biocompatibility

of chemically synthesized poly(GVGVP) was

already known was there adequate impetus

to purify microbially prepared (GVGVP)25i.

Otherwise, it would have been presumed, as

had been widely expected, that the toxicity of

inadequately purified (GVGVP)25i was an

inherent property of the protein-based

polymer. To be left in such a state of misunder-

standing would have meant that the dramatic

potential of elastic protein-based polymers for

use in medical appHcations would be neither

appreciated nor realized. The inflammatory

response ehcited by an inadequately purified

biosynthetic elastic protein-based polymer

would have overwhelmed most considered

medical applications.

Our research utilizing chemical synthesis of

repeating peptide sequences is represented

in over 170 scientific publications. It utilized

classic solution synthesis and to a much lesser

extent solid phase methods. The primary focus

in all cases has been development of an under-

standing relevant to structure, function, and

mechanism rather than development of syn-

thetic methodologies, although some of the

latter did indeed occur principally due to the

expert capacities of T. Ohnishi, K. Okamoto,

R. Rapaka, K.U. Prasad, T.R Parker, and D.C.

Gowda. Here we note a few issues relevant to

the production of protein-based polymers, that

is,

of polymers composed of repeating peptide

sequences.

9.2.2.2 Classic Solution Methods

for Chemical Synthesis of

Repeating Sequences

A principal concern during chemical synthesis

is racemization, the formation of some amino

acid residues of the D-configuration rather

than maintaining only the L-configuration. The

result of only L-amino acid residues in the

expressed protein, of course, results from

protein synthesis by means of the genetic code

and recombinant DNA technology. (See Figure

3.3 and the associated text in Chapter 3 to

review the mirror image relationship between

L- and D-amino acid residues and to appreciate

the consequences relating to structure and

function.) In particular, during the chemical

activation of the carboxyl group of an amino

acid and its subsequent reaction with an amino

function of another amino acid, the amino acid

residue with the activated carboxyl can racem-

ize;

some D-amino acid residues become

incorporated into the growing chain with a

consequence of structural disruption like that

schematically represented in Figure 3.3C.

There are two ways to avoid recemization

during chemical synthesis. One way is to rely at

critical steps on the glycine (Gly, G) residue,

which with two hydrogens on the a-carbon

does not form mirror images. Therefore, for G,

there is no consequence of interchanging posi-

tions of the a-carbon substituents during acti-

vation and reaction. The second way is to rely

on the prohne (Pro, P) residue in which the

bridging of the R-group, -CH2-CH2-CH2-,

between a-carbon and nitrogen atom of the

same residue limits racemization. Accordingly,

with repeating units containing G and P, which

are critical residues in the repeating sequences

of elastic protein-based polymers, the strategy

is to build the repeating unit with a G or P at

its carboxyl terminus and to utilize crystalliza-

tion to remove component peptide sequences

that contain racemized residues.

The build-up strategy for the GVGVP or

VPGVG repeating unit is a 2 x 3 strategy. First

the dipeptide VP is synthesized and crystallized

to form pure dimer containing only L-amino

acid residues. Then the GVG tripeptide is syn-

thesized and also crystallized to result in pure

GVG containing only L-valine (Val, V). The

dipeptide can be added to the tripeptide to

form VPGVG, or the tripeptide can be added

to the dipeptide to form the pentamer GVGVP.

Both pentapeptides have either the required

G or the required P to prevent racemization

478

9. Advanced Materials for the Future

during polymerization of the pentamer to make

the poly(GVGVP) or poly(VPGVG). Because

higher molecular weight peptides could be

obtained using GVGVP, this became the poly-

merization of preference. Also a search for the

best activating reagent resulted in molecular

weights higher than had been previously

achieved by chemical synthesis."*^'^^

9.2.2.3 Merrifield Solid Phase Methods

for Chemical Synthesis of

Repeating Sequences

The Merrifield solid phase synthesis

approach'*'^^ constitutes an apparatus that is

constructed and programmed to go through the

many many steps involved in forming a single

peptide bond and to grow the peptide chain

by coupUng each desired amino acid of the

sequence to the growing chain attached to a

solid phase, a so-called resin. No matter how

much care may be taken at each coupUng step,

however, there remains a finite probability of

racemization. For protein-based polymers this

Umitation is overcome by proceeding with

pentamer additions, by adding GVGVP or

VPGVG, for example. This has the great advan-

tages of preparing racemization-free polymers

of essentially a single chain size and of setting

up and programming the apparatus to run con-

tinuously through many couplings."^^ There is

the disadvantage, however, because coupling

efficiency is not 100%, that much pentamer is

required for the synthesis and is not incorpo-

rated into the growing chain.

The advent of recombinant DNA technology,

two decades after the remarkable Merrifield

advance, has overtaken many of the advantages

of the Merrifield approach. After the time-

consuming gene construction and development

of a good expression system, large quantities of

elastic protein-based polymers are produced in

a short time, as shown in Figure 9.5.

9.2.2.4 Practicality of Chemical Synthesis

A major limitation of chemical synthesis is cost,

which is central to the practicality of particular

applications. Cost of production at the labora-

tory bench is on the order of $200/gram. Scale

up could be expected to reduce that cost by an

order of magnitude. For medical applications

that require costly FDA approval, however,

there is the added cost of what is called good

manufacturing practices, which increase costs

severalfold. Accordingly, any product that

would not be competitive at $100/gram would

be challenging to consider by chemical synthe-

sis of protein-based materials, particularly

when there can be strict functional require-

ments for certain elements of purity, optical and

otherwise.

Nonetheless, when carried out with sufficient

care,

chemical synthesis of the polymers can be

the best approach for establishing biocompati-

bility of protein-based polymers. As discussed

below, the chemically synthesized products that

have been carefully purified became primary

standards against which purification of biopro-

duced polymers could be held. The specific

disadvantages of chemical synthesis are its

expense, the inability to be totally free of

racemization, its reproducibility, and the need

to use noxious and toxic chemicals. On the

other hand, the primary challenge of biosyn-

thesis using recombinant DNA technology is

one of removing the toxic components unique

to the producing organism or plant.

9.2.2.5 Biocompatibility of Chemically

Synthesized Protein-based Materials

The biocompatibiUties of the three physical

states of these protein-based polymers, that

is,

hydrogel, elastic, and plastic, were ini-

tially determined using the representative

chemically synthesized products. The polymer

poly(GGAP) may be considered the parent

polymer for the hydrogel state, because it exists

as a hydrogel except under extreme conditions

of high concentrations of multivalent salts.

Poly(GVGVP) is the parent polymer for the

elastomeric state, as it derived from the mam-

malian elastic fiber. It was the beginning

sequence from which many of the other poly-

mers were derived, initially by design and

chemical synthesis of compositional variants.

Poly(AVGVP) was the original sequence for

the plastic state; it derived by simple substitu-

tion of a glycine (G) residue of poly(GVGVP)

by an alanine (A) residue. The initial purpose

9.2 Production of Protein-based Polymers

479

of the synthesis of poly(AVGVP) was to test

the newly proposed basis for near ideal or

entropic elasticity.

Of course, the primary requirement for use

of these polymers as part or all of a medical

device is that the protein-based polymer must

be sufficiently nontoxic, that is, it must exhibit

adequate biocompatibility. As representative

polymers for each of the interesting physical

states,

each of the above three compositions

has been thoroughly examined by the standard

set of 11 tests recommended by the American

Society for the Testing of Materials (ASTM) for

materials in contact with tissue, tissue fluids,

and blood.

9.2.2.5.1 Perspective from a Battery of

ASTM Tests

According to the ASTM 1987 Standard Prac-

tice for Selecting Generic Biological Test

Methods for Materials and Devices (Designa-

tion F784-87), there are nine tests for materials

in contact with tissue and tissue

fluids.

An addi-

tional two tests are added for materials to be

used in contact with blood. One of the nine

tests,

the test for carcinogenicity, is unnecessary

if the mutagenicity test is negative. As shown

in Table 9.3^^ for poly(GGAP), and as is also

the case for poly(GVGVP), these polymers

would appear to be even less mutagenic than

the saline negative control. Accordingly, no

carcinogenicity testing is required for these

polymers, whereas a positive control, dexon, a

suture material with a high rate of mutagenic-

ity, presumably did require carcinogenicity

testing but, as it is in use, it must be of suffi-

ciently low carcinogenicity that it can be used.

Because antigenicity and immunogenicity

are such potential issues for protein-based

polymers, both the systemic antigenicity, as

determined by the British Pharmacopoeia

TABLE

9.3. Plate incorporation assay for the Ames mutagenicity test for X^°-poly(GGAP).^

Salmonella typhimurium

tester

strains

Saline

control)

Saline

test

article

solution

(undiluted)

Saline

w/S-9 (-

control)

Saline

w/S-9

test

article

solution

(undiluted)

Dexon,

mg/ml

control)

Dexon,

mg/ml

w/S-9

control)

Sodium

azide, 0.1

mg/ml

control)

Sodium

azide, 0.1

mg/ml

w/S-9 (+

control)

2-Nitrofluorene,

mg/ml

control)

2-Nitrofluorene

w/S-9

(-1-

control)

2-Aminofluorene,

0.1

mg/ml

control)

2-Aminofluorene

w/S-9

("f-

control)

TA98

1,048

1,256

N/A

TAIOO

180

172

173

180

1,408

1,048

N/A

184

1,296

Number

revertant

colonies

(agerage

duplicate

plates)

TA1535

TA1537

N/A

2,752

3,176

N/A

247

1,048

N/A

TA1538

N/A

2,800

2,240

2,816

* Test article: X^°-poly(GGAP), batch CG65PA. In no case was there a twofold or greater increase in the reversion rate

of the tester strains in the presence of the test article solution. N/A = not applicable.

^ Reports from North American Science Associates (NamSA®).

Source: Reproduced with permission from Urry et al.^^

480 9. Advanced Materials for the Future

TABLE

9.4. Guinea pig sensitization dermal reac-

tions—challenge with X^°-poly(GGAP), batch

CG65PA.^

Animal

(No.,

Group)

Test

Control

Hours

0.5

following patch removal

0.5

^ Test article solution was received in the right flank.

Source: Reproduced with permission from Urry et al.^-

Antigenicity Test (BPAT), and the dermal sen-

sitization study (a maximization method) were

used. The scoring for the dermal sensitization

study, shown in Table 9.4^^ for poly(GGAP),

is the same as in Table 9.5^^ for the intracuta-

neous toxicity study. In the latter case, there

simply

neither erythema (redness) nor edema

(swelling) due to injection of the polymer. In the

former case, the test polymer was as innocuous

as the negative control of salt water. Obviously,

these polymers are remarkably biocompatible.

The resulting 11 tests are summarized in Table

9.6"^^

for poly(GVGVP). An even more stringent

examination for poly(GVGVP) is discussed

below, but first we consider poly(AVGVP).

The summary of the 11 tests for poly

(AVGVP) (see Table

in Urry et al."') reads the

same as for poly(GGAP) and poly(GVGVP).

A direct comparison of the guinea pig dermal

sensitization test for poly(AVGVP) in Table

9.7, however, shows poly(AVGVP), although

yet considered nonsensitizing, to be more

reactive than that shown in Table 9.4 for

poly(GGAP) and that for poly(GVGVP) (see

Table 6 in Urry et

al."^^).

TTiis in part demon-

strates the truly remarkable biocompatibility of

poly(GVGVP) and poly(GGAP).

9.2.2.5.2 Refractory to Formation of

Monoclonal Antibodies

Because of the need for monoclonal antibodies

to poly(GVGVP) in order to follow biopro-

duction of this polymer in the tobacco plant,

truly exceptional efforts were undertaken to

achieve antibodies. Attempts to make mono-

clonal antibodies directly to poly(GVGVP)

were simply unsuccessful despite repeated

efforts using the most sensitive and repeatedly

sensitized mice and rats. It was possible to

find three weak monoclonal antibodies to

poly(AVGVP) that had exhibited good bio-

compatibility^^ but not the same extraordinary

biocompatibility as for the representatives

of the hydrogel, poly(GGAP), and elastic.

TABLE

9.5. U.S. Pharmocopeia intracutaneous toxicity observations for

poly(GGAP)

Rabbit

No.

69235

Test

Control

69226

Test

Control

Hours

ER ED

0 0

Reports from North American Science Associates (NamSA). Date prepared, 2-

8-93;

date injected,

2-8-93;

date terminated,

2-11-93.

Erythema (ER): 0 = none, 1

= barely perceptible,

= well defined,

= moderate, 4 = severe. Edema (ED): 0 =

none,

= barely perceptible, 2 = well defined, 3 = raised

mm,

4 = raised

mm.

Rating (test and control): 0.0-0.5, acceptable; 0.6-1.0, slight; >1.0, significant.

Source: Reproduced with permission from Urry et al.^^

9.2 Production of Protein-based Polymers

481

TABLE

9.6. Summary of 11 biological test results for poly(VPGVG) and its cross-linked matrix.^

Test

Ames (mutagenicity)

Cytoxicity

Systemic toxicity

Intracutaneous toxicity

Muscle implantation

Intraperitoneal

implantation

Systemic antigenicity

(BPAT)

Sensitization

(Kligman test)

Pyrogenicity

Clotting study

Hemolysis

Description

Determine reversion rate

wild type

histidine-dependent mutants

Agarose overlay determines cell death

and

zone

lysis

Evaluate acute systemic toxicity from

intravenous

intraperitoneal injection

Evaluate local dermal irritant

toxic effects

by injection

Effect

living muscle tissue

Evaluate potential systemic toxicity

Evaluate general toxicology

Dermal sensitization potential

Determine febrile reaction

Whole blood clotting times

Level

hemolysis

in the

blood

Test System

Salmonella

typhimurium

L-929

mouse

fibroblast

Mice

Rabbit

Rat

Guinea pigs

Rabbit

Dog

Rabbit blood

Results

Nonmutagenic

Nontoxic

Favorable

Nonantigenic

Nonsensitizing

Nonpyrogenic

Normal clotting time

Nonhemolytic

Reports from the North American Science Associates (NAmSA).

Source: Reproduced with permission from Urry et al."^^

poly(GVGVP),

states.

Fortunately,

one

of the

weak monoclonal antibodies

poly(AVGVP)

was

found

cross-react,

more weakly

yet,

with

poly(GVGVP) such that poly(GVGVP) could

identified

in the

transgenic tobacco

plant.

Further efforts

utilize

the

monoclonal

anti-

body

poly(AVGVP)

for

ready identification

poly(GVGVP)

in the

development

micro-

bial expressions systems were abandoned

due

inadequate

reactivity.

TABLE

9.7. Guinea pig sensitization dermal reactions—Challenge test article: X^°-poly(AVGVP), batches

CG20WR and CG156WR.

Animal

No./Group

1 Test

2 Test

3 Test

4 Test

5 Test

6 Test

7 Test

8 Test

9 Test

10 Test

11 Control

12 Control

13 Control

14 Control

15 Control

Site

0.5

SiteB

0.5

Site

0.5

Hours following patch removal

SiteB

0.5

Site

0.5

SiteB

0.5

Site

0.5

SiteB

0.5

Site A = left flank = SC control vehicle; site B = right flank = SC test extract. Reports from North American Science

Associates (NAmSA®).

Source: Reproduced with permission from Urry et al."^^

482

9. Advanced Materials for the Future

9.2.2.5.3 Response of Human Monocytes to

Elastic Protein-based Polymers

There is one further telling study that bears

on the nature of the interaction, or absence

thereof,

of poly(GVGVP) with human cells,

monocytes, whose role it is to identify and

destroy foreign materials in the body. Grace

Picciolo and coworkers at the Center for

Devices and Radiological Health of the FDA,

using instrumentation designed to evaluate the

reaction of human monocytes to biomaterials,

found poly(GVGVP) and the related poly-

mer containing cell attachment sequences

poly[40(GVGVP),(GRGDSP)] to subdue the

background activity of the monocytes.^^ This

innocuous, and possibly even passivating,

response of human monocytes to the basic

polymer poly(GVGVP) means that it becomes

possible to add different biological activities

very selectively. The capacity will be to elicit

selectively the desired biological response to

the added sequence without having to tolerate

background activities of the carrier polymer.

This becomes especially useful when the carrier

protein-based material has an elasticity that can

match that of the natural tissues. One of the

results of this combination of biocompatibility

and biological elasticity is a group of appUca-

tions referred to below as soft tissue restoration.

First, however, the importance of purification

from E. coli fermentation is emphasized, as this

is necessary before the remarkable biocompat-

ibility of elastic protein-based polymers can be

utilized.

9.3 Purification of Tj-type

Protein-based Polymers

By Tftype, we mean polymers that exhibit

inverse temperature transitions in which the

protein-based polymers hydrophobically asso-

ciate on raising the temperature. Tt represents

the onset temperature for the transition. For

the elastic-contractile model proteins of inter-

est here, the inverse temperature transition is

seen as a phase separation resulting from both

intermolecular and intramolecular hydropho-

bic association.^^ On raising the temperature

above that of the onset temperature for the

transition, Tj, Tt-type protein-based polymers

go from being dissolved in solution to being

separated from solution. There exists another

very important means of achieving the phase

separation of Tt-type protein-based polymers;

it is to lower the temperature for the onset of

the phase transition, Tt, from above to below

the operating temperature. When concerned

with the physiological operating temperature of

37° C, to lower the value of Tt from 3T to 25° C

causes the Tt-type protein-based polymer to

separate out of solution. As treated extensively

in Chapter 5, the process can be one of

self-

assembly by hydrophobic association.

As demonstrated below, this property, funda-

mental to the diverse protein functions dis-

cussed in Chapters 5,7, and 8, provides the basis

for purification.^^

9.3.1 Purification by Phase Separation

(Inverse Temperature Transition)

The purpose below is not to describe method-

ologies for evaluating purity, but rather it is

to use selected methodologies to demonstrate or

to monitor the effectiveness of purification

based on phase separation.The utilized method-

ologies are SDS PAGE (sodium dodecyl

sulfate polyacrylamide gel electrophoresis) and

radiolabeling of contaminants. The lysed or

ruptured cells of E. coli, transformed to produce

the specific Tftype protein-based polymer,

provide the impure reference state against

which subsequent purification steps may be

compared.

93.1.1 SDS PAGE Demonstration of

Purification by Phase Separation

Figure 9.6 represents a negative staining tech-

nique involving a CUCI2 stained gel to demon-

strate purification at different steps and for

different fractions during the purification by

phase separation of (GVGVP)i4i. Lane 1

contains all of the components of the gross

lysed cells of E. coli transformed to produce

(GVGVP)i4i; as such it shows all of the protein

bands of the transformed E. coli. As will be dis-

cussed, the bulging band of lane 1 is the product

9.3 Purification

Trtype Protein-based Polymers

483

FIGURE

9.6. CuCl2-stained gel of SDS

PAGE shows purification

(GVGVP)25i

by phase separation. (Lane

Lyse-

transformed cell showing

all E.

coli

proteins plus the bulging band

product,

(GVGVP)25i. (Lane 2) Residue

the cold

spin where

the

protein-based polymer

plus most E.

coli

protein remained in solu-

tion. (Lane

Supernatant

37°C warm

spin that contains essentially

all of the

remaining E.

coli

protein. (Lane 4) Model

protein, (GVGVP)25i, phase separated

at 37°C from

the E.

coli protein that

remained

solution (lane

3).

(Repro-

duced with permission from Urry

al.^^)

Mol. Wt.

97,400

68,000

43,000

29,000

(GVGVP)i4i. The sample

then chilled

to ensure dissolution

of the

protein-based

polymer

and

then centrifuged;

the

residue,

which contains insoluble cell debris, embedded

proteins,

and any

remaining nonlysed cells,

shown

lane

2. The

supernatant

of the

cold

spin

heated

37°

C and

again centrifuged.

The supernatant

of the

warm spin

lane

shows

the

panoply

E, coli proteins. When

the

precipitate

of the

warm spin

redissolved

and

run

lane

4, a

single band

purified product,

(GVGVP)i4i,

observed. This purification

achieved entirely

phase separation.

9.3.1.2 Purification

Phase Separation

Shown

Carbon-14 Labeled

coli

The effectiveness

phase separation

purifi-

cation

of the

elastic protein-based polymer

(GVGVP)25i

can be

evaluated

carbon-14

labeling

all of the E.

coli constituents except

the protein-based polymer. Incubation

of E.

coli with

the

radiolabeled carbon source

C-14

glucose enables this.

coli, labeled

this

means, is lysed and added

the lysed cells from

a fermentation

transformed

coli

in the

absence

carbon-14,

and the

mixture

puri-

fied. In this

way

only

the E.

coli impurities

are

labeled.

Figure

9.7

demonstrates

the

reduction

impurities with each phase separation cycle.

short, three cycles reduce impurities

over

factor

1,000.

Using

Img of

polymer, three

cycles

phase separation decrease impurities

from

1,428,600

parts

per

billion

(ppb) to

1,365

ppb.^^

discussed below, further purifi-

cation lowers impurities,

judged

Western

immunoblot techniques,

less than

ppb.

9.3.2 Physical Characterization

and

Verification

Product Integrity

9.3.2.1 Gross Visualization of the Phase

Separated Product

An example

(GVGVP)25i

as the

phase sepa-

rated product

shown

Figure

9.5 and

dis-

cussed above

section 9.2. By the small change

of adding

one CH2

group

per

pentamer,

the

gross physical properties change substantially.

484

9. Advanced Materials for the Future

Retention of C-14 Impurities in Polymer

Purification

1.60E+08

1.40E+08

1.20E+08

S l.OOE+08

0 8.00E+07

6.00E+07

4.00E+07

2.00E+07

O.OOE+00

H—1—^^M—1—liiMiai—1 1 1 1 1

Cell Sup. C7cla-1 Cycle-2 Cycle-a Cycle-4 Cycle-S Dialysis Additional

Step

Fractions

FIGURE

9.7.

Purification of

(GVGVP)25i,

followed by imately 10-fold. (Unpublished data, of

Pattanaik,

loss of carbon-14-labeled E. coli constituents as a T.M. Parker, and D.W. Urry. Courtesy of Bioelastics

function of each cycle of phase separation. Each Research, Ltd.)

phase separation cycle reduces impurities by approx-

As shown in Figure 9.8, the phase separated

state of a relatively small amount of protein-

based polymer, (GVGIP)26o, looks like a small

pancake that can be pulled into a tough band 1

meter in length. The elastic protein-based poly-

mers in Figures 9.5 and 9.8 are fundamental

compositions that can be treated in many

dif-

ferent ways to result in products of commerciar

interest. With designed modifications in the

compositions, many more specialized appUca-

tions result, as is but briefly noted as this

chapter progresses.

9.3.2.2 Nuclear Magnetic Resonance

Evaluation of Sequence Integrity

and Purity

The sequence and purity of the basic repeating

30mer of Model Protein v of Table 5.5 are ver-

ified by one- and two-dimensional nuclear mag-

netic resonance (NMR) spectroscopy.^"^ In

particular in Figure 9.9, by a combination of

the one-dimensional spectrum at the top of

the figure and the two-dimensional HOHAHA

map in the upper left, the through-bond

proton-proton couplings are dissected and

assignments made.

TTiis

result demonstrates the

absence of unexpected amino acid residues in

the repeating 30 residue sequence.

At the lower right of the two-dimensional

map,

nuclear Overhauser enhancement spec-

troscopy (NOESY) demonstrates a set of

through space proton-proton contacts that

allow determination of sequence. As Usted at

the top of Figure 9.10, there are four types of

through space proton-proton contacts. Contact

between the aCH proton of residue i and the

NH proton of residue i + 1 is defined as dox and