EP0964918A2 - Reversibly inactive synthetic beta-galactosidase - Google Patents

Abstract

A synthetic β-galactosidase enzyme which differs from naturally-occuring β-galactosidase in that said synthetic enzyme is substantially inactive when present in packaged milk, and is activated by a chemical or condition naturally present in the gastrointestinal tract of humans or during processing of the milk for example during cheese making.

Description

β-GALACTOSIDASE WITH REVERSIBLY INACTIVE LACTASE ACTIVITY

BACKGROUND OF THE INVENTION

Lactose is one of the main milk components and it is found exclusively in dairy

products and foods incorporating them. Its presence is necessary for normal lactation.

Lactose cannot be absorbed directly by the intestine. Rather, it must be cleaved into its

constituent monosaccharides by the naturally occuring enzyme lactase-phlorizin hydrolase

(β-galactosidase). This enzyme is attached to the glycocalyx or lining of the small intestine

and in our gut microflora. All infant mammals, including humans, express intestinal

β-galactosidase in high concentrations. However, as we age, the amount and activity of this

enzyme gradually decreases and lactose digestion is impaired. This condition is termed

"lactose intolerance", and is characterized by a number of gastrointestinal disorders when the

individual consumes milk or other non-fermented dairy products. Lactase deficiency linked to

lactose malabsorption and milk intolerance affects the majority of the world's population.

Several food-grade β-galactosidase enzyme preparations from various organisms have been

used to prehydrolyze lactose before milk consumption or administered orally as "enzyme

replacement therapy" in order to eliminate lactose malabsorption. The products of this

hydrolysis, glucose and galactose, are considerably sweeter and found in equimolar

concentrations as lactose. This results in a considerably sweeter product. This increased

sweetness has been identified as an unacceptable flavour attribute of lactose-hydro lyzed

products in consumer test panels. SUMMARY OF THE INVENTION

The present invention features the engineering of a β-galactosidase protein

(prolactase) with specific performance attributes, i.e. to be reversibly inactive during regular

storage conditions of the milk and to function (hydro lyze lactose) within the human

alimentary track. The invention will reconcile the need for milk with "normal" organoleptic

properties while at the same time hydrolyzing the milk lactose, within the stomach or small

intestine upon ingestion (post-taste buds lactose hydrolysis).

Within the gastrointestinal tract two very different environments exist: the gastric

environment, which has an acidic pH (2-5), and the intestinal lumen, which is neutralized by

the pancreatic secretions, β-galactosidase genes are available that encode enzymes which

would function in various environments. For example, the pH optimum of Aspergillus niger

β-galactosidase is 3-4, that of Aspergillus oryzae is about 5.0, while that oϊK. lactis is

6.5-7.5.

The time parameter in the stomach is governed by the rate of flow of the chyme

through the system. In the stomach the volume of the meal is the predominant regulator of

the rate of gastric emptying. For example, 330 ml of milk would be cleared in 80 minutes,

while 1,250 ml milk would require 140 minutes. The most conservative case, consumption

of a glass of milk, would provide a window of gastric hydrolysis of 80 minutes. However,

since faster empting times are reported, a safe window would be 30 min. Furthermore, the

rate by which lactose for example is moving from the gastric environment to the intestine

may affect the rate of hydrolysis. Time would be less of an important factor should the

enzyme of interest function in the intestine or have residual activity in the intestine. Thus, the invention provides a synthetic β-galactosidase enzyme which differs from

naturally-occurring β-galactosidase in that the synthetic enzyme is substantially inactive

when present in milk, and is activated by a chemical or condition naturally present in the

gastrointestinal tract of humans. Preferably, the β-galactosidase portion of the synthetic

enzyme is linked to peptide or polypeptide (which can be thought of as a "regulatory

domain") via a cleavage site (preferably an enzyme-cleavable peptide bond). The role of the

regulatory domain is to reversibly inactivate/suppress the hydrolyzing activity of

β-galactosidase; the cleaving enzyme is one which is present preferably in the human

gastrointestinal tract, but substantially absent from milk; the cleavage site should be one

which is substantially and not cleavable by any other chemicals or conditions found in milk.

The invention also features DNA molecules encoding the synthetic enzymes of the

invention, and recombinant cells containing those DNA molecules.

The invention also features transgenic mammals whose mammary glands express the

DNA molecule encoding the synthetic enzyme; the DNA molecule in this case includes a

mammary-specific promoter controlling transcription of the DNA encoding the enzyme, and

also preferably includes a nucleic acid sequence encoding a signal peptide which causes

secretion of the enzyme into the milk of the transgenic mammal.

DETAILED DESCRIPTION

The drawings are first briefly described.

Brief Description of the Drawings

Fig. 1 is a diagrammatic representation of the construction of a plasmid used as the

expression vector for a synthetic enzyme of the invention.

Fig. 2 is a diagrammatic representation of the construction of a vector containing A. niger β-galactosidase-encoding DNA under the control of the α-Gla (α-glucoamylase)

promoter.

Fig. 3 is a diagrammatic representation of the construction of a vector containing

DNA encoding A. niger β-galactosidase, fused to a bovine lactoferrin tail.

Fig. 4 is a diagrammatic representation of the construction of a vector containing

DNA encoding A. niger β-galactosidase fused to a polyamino acid tail-encoding sequence.

Fig. 5 is a diagrammatic representation of the construction of a vector containing a

DNA encoding A. niger β-galactosidase fused to a sequence encoding a bovine α-

lactalbumin tail. Fig. 6 is a diagrammatic representation of an expression cassette in which the A. niger

β-galactosidase gene is fused to A. niger β-galactosidase cDNA (tail) through a pepsin

cleavage site under the control of an niger glucoamylase gene promoter.

Fig. 7 is a diagrammatic representation of a vector in which the 3' end of the

β-galactosidase gene is modified to encode a protein with a cleavage site recognizable by

pepsin.

Fig. 8 is a diagrammatic representation of the fusion of the modified β-galactosidase

with the β-galactosidase cDNA shown in Fig. 7 into a second vector.

Fig. 9 is a diagrammatic representation of the strategy of the construction of a vector

containing genomic A. niger β-galactosidase-encoding DNA fused through a pepsin site to

A. niger β-galactosidase cDNA (tail).

Fig. 10 is a diagrammatic representation of the overall strategy for constructing a

vector containing A. niger β-galactosidase-encoding DNA, under the control of the A. niger glucoamylase promoter, with a bovine casein tail fused to β-galactosidase through a pepsin-

cleavable site.

Choice of β-galactosidase

The enzyme to be used must be acid resistant and resist denaturation within the

stomach from pH 1.0 (fasting state) to 5.0. The enzyme must retain activity in the presence of

proteolytic enzymes, pepsin in the stomach and the pancreatic proteases in the intestine

lumen.

The β-galactosidase of A. niger has a pH optimum in the range of 3-5, which makes it

suitable for processing of acid whey and its permeate. This enzyme has relatively high

temperature optima and is typically used at temperatures up to 50° C with considerable

stability. The attributes of the enzyme make it also suitable to function in the gastric

environment, and together with A. oryzae β-galactosidase has been used to alleviate the

symptoms of lactose malabsorption in the form of commercially available tablets taken just

before the consumption of a dairy meal. The β-galactosidase of A. niger is a suitable

candidate because it retains activity after incubation in gastric juice. The β-galactosidase gene

of A. Niger is described in Huang et al. U.S. Patent Application Serial No. 08/551,459;

hereby incorporated by reference.

Engineering a lactase with a prosequence

Numerous enzymes and hormones in nature have as part of their structure a peptide

which serves a regulatory purpose. When the pro-peptide is attached to the rest of the

protein, it may alter the protein folding or block an active site. When these "pro" or "pre"

peptides are removed, the enzyme becomes active. Examples of this type of regulation are

common within the digestive system, eg. gastric or intestinal proteases. The evolution of this type of regulation of enzymatic activity has permitted gastric and intestinal cells to synthesize

powerful proteolytic enzymes without risk of auto-degradation. The "pro" form of the

enzyme is completely inactive. Following secretion from the cell, the pro-enzyme is

activated by the removal of the "pro" regulating peptide. Damage to the exterior of the cells

is prevented by a thick mucous layer which is impervious to proteolytic action.

Gastrointestinal protease structure and function are well understood. For example, the target

sequences for cleavage are known and the molecular details of cleavage dynamics are known.

Pepsin is found in significant quantities within the gastrointestinal system. Its presence is a

prerequisite for dietary protein digestion. Lactose hydrolysis will occur inside the human

body, where the relatively high temperature of 37 °C will speed reactions.

The concept of activating enzymes as a result of an acidic pH shift and/or proteolytic

cleavage has been described as a regulatory method for both endogenous and foreign

enzymes.

The work by Brem et al (1995) shows the activation of chymosin. Their recombinant DNA

construct involved a bovine casein promoter driving the expression of bovine chymosin

within a transgenic rabbit system. Chymosin was synthesized as a prochymosin which is

activated into the active enzyme via the action of gastric pH and/or proteases. Specifically,

exposure to 0.5 N HC1, pH=2.5 activated the enzyme.

Genetic Engineering of Tails

Recombinant DNA techniques have been used to add fusion tails to various proteins

in order to facilitate their purification. Fusion-tail systems have been developed using

His-tags, streptavidin binding peptides, IgG binding domains, glutathione S-transferase and

maltose binding protein for affinity chromatography (Ford et al, 1991. Protein Expression and Purification 2:95-107). Genetic fusions of E. coli β-galactosidase with charged

polyamino acid tails have been generated in order to facilitate their purification (Niederauer

et al., 1994). In these examples, inactivation of the protein of interest by the addition of the

fusion-tail to be used as an affinity/purification handle is an undesirable effect.

In contrast, in the present invention, the fusion of the regulatory tail to a

β-galactosidase, for example, through an enzyme recognition/cleavage recognition site,

desirably renders the new hybrid molecule reversibly inactive. Under appropriate conditions

this site is recognized by a specific protease and releases the β-galactosidase molecule which

hydrolyzes lactose. Nature of Cleavage Sequence

The cleavage sequence is engineered for recognition by a proteolytic enzyme. In

Table 1 below, a partial list of such proteolytic enzymes and their target sequences are

presented.

Table 1 : Endopeptidase cleavage sites to be used in the construction

of the prolactase molecule GI Site Enzyme present Recognition site Specificity

Stomach pepsm N side of Rn Rn=Leu, Asp, Glu, Phe,

Tyr, Trp

gelatinase Gly-Pro-Glu-Gly-Ile- Ala-Gly-His

Intestine trypsin C side of Rn Rn=Lys, Arg chymotrypsin C side of Rn=Tyr, Phe, Trp,

Leu elastase proelastase carboxypeptidase N side of C terminus Rn no Arg, Lys, Pro enterokinase protrypsm

Pro-β-galactosidase Design

The expression vectors to be used to produce the prolactase enzyme are presented in

more detail in the examples given below. Generally, the constructs include a promoter region

that allows a high level of expression, a secretion signal, the prolactase, termination

sequences, antibiotic resistant genes (for example amp) for propagating the construct in

bacteria, and an origin of replication. The choice of promoter, termination and signal

sequences depends on the host organism to be used as the expression system. These components are addressed below:

1. The signal peptide can be one from the endogenous β-galactosidase gene if it is a

naturally secreted (i.e. A. niger or A. oryzae), or other signal peptides can be used which have

proven to be effective in the system, in which the recombinant prolactase is going to be

produced (i.e. from the A. niger glucoamylase gene). If the prolactase is to be produced

through, for example, transgenic animals, one may use the signal peptides from the casein or

whey family proteins in order to achieve a vectorial secretion of the enzyme from the

mammary epithelial cells into milk. Such systems are described, e.g., in Gordon et al., 1987,

Biotechnology 5:1103-1187; and Hennighausen, 1992, J. Cellular Biochemistry 49:325-332,

hereby incorporated by reference.

2. The β-galactosidase, e.g., from A. niger or A. oryzae, has desirable functional

properties (pH optimum of 2.5-5.0) and lactose hydro lyzing activity.

3. Regulatory domain. The fused gene can be engineered to include a cleavage site,

e.g., as described in Table 1, and a blocking domain, or "tail," which will suppress enzymatic

activity until it is removed by the action of a specific enzyme recognizing the cleavage site.

The engineered tail will render the β-galactosidase molecule reversibly inactive due to

misfolding or due to blockage of the active site. Native folding of the β-galactosidase will

be resumed upon cleavage of the tail by the appropriate enzyme. Tails with variable lengths

can be engineered in order to identify the one rendering the molecule inactive. This "tail"

may also serve as an affinity handle during purification of the enzyme from culture broth.

The tail domain may be added at the 3'-, 5'- or both ends of the gene whose activity is

to be modulated. Proteins of known length and function which are normal constituents of milk such as caseins, lactoferrin or lactalbumin can be used as tails. Alternatively, smaller

polyaminoacid tails can be used that add either a positive or negative charge to the

β-galactosidase molecule. Variations or truncated versions of the tail can also be fused in a

matter similar to the full-length tail. Such truncated versions of the tail can be generated for

example by the action of Exonuclease III. Truncation can be either at the amino or carboxyl

termini of the tail of interest.

The prolactase molecules (with the tail part having a series of deletions) are

introduced into the appropriate host expression system. Transformants and media from

cultures carrying these constructs are first screened for successful expression by the use

of antibodies. The antibodies used can recognize either the β-galactosidase or the

tail part of the prolactase molecule. It is preferable that the host to be used as an expression

system not have any endogenous β-galactosidase activity which may interfere with the newly

expressed recombinant prolactase molecule. A preferred fungal host is for example the one

with the endogenous

β-galactosidase gene disrupted. Such a host is described in the Huang et al. patent

application USSN 08/551,459, filed November 1, 1995, supra. When successful expression

of the prolactase molecule is confirmed by Western blotting then the media is further tested

for the newly expressed prolactase protein.

4. Cleavage Sequence

The cleavage sequence is engineered to act as a recognition site for the specific

cleaving enzyme. Table 1 provides a partial list of such proteolytic enzymes and their target

sequences.

In addition specific cleavage sites can be added for a specific application. For example, activation of the prolactase in the gastric environment would require a site that is recognized

by pepsin or one that is activated by the low acidic pH. The addition of κ-casein as a

regulatory domain can generate a fusion molecule that could be added in milk during the

cheese manufacturing processing. It can be engineered so that the cleavage site is recognized

by the enzyme chymosin/renin so that upon addition of chymosin the site is cleaved (Phe-

Met), the tail is removed, and the β-galactosidase is released, thereby hydrolyzing the lactose

present in the whey waste.

Prolactase Production

Once the genetic engineering of the protein has been accomplished, it should be

producible in large quantities by a cost effective means. Fermentation techniques are

currently used on an industrial scale for the production of many food enzymes (chymosin,

β-galactosidase). The organism selected as the production vehicle will depend on several

process parameters asssociated with the design of the DNA construct. A variety of organisms

can serve as production vehicles. Prokaryotic fermentation typically utilizes Escherichia coli

(Emtage et al, 1983; Zhang et al, 1991) or Bacillus subtilis (Parente et al, 1991) or a variety

of GRAS microorganisms. For more biochemically complex molecules, the yeast,

Saccharomyces cerevisiae (Goff et al, 1984) is routinely used. Fungal fermentation also

provides an efficient expression system for the production of recombinant proteins.

EXAMPLE 1

Prolactase Expression System

Expression vector

The expression vector was derived from the pGla expression vector kindly provided

by Dr. R. Storms (Concordia University, Montreal) with the following modification (Figure 1). The pUC18 backbone of the original pGla vector was replaced with a shorter pUC18

backbone lacking the LacZ portion. This modification was made in order to avoid the

possibility that any activity from the LacZ product interferes with the activity of the

expressed beta-galactosidase. The following steps were performed: The pGla vector (Figure

1, step 1) was first digested with Sal restriction enzyme (Promega, Madison, WI, USA) and

re-circularized using T4 DNA ligase (Pharmacia Biotech, Baie D'Urfe, Quebec, Canada) in

order to remove one of the Sail and Sad restriction sites. The resulting plasmid was digested

with Hindlll (Pharmacia) and Sad (Pharmacia), treated with T4 DNA polymerase

(Pharmacia) in the presence of dNTPs (Pharmacia) and purified from agarose gel. The band

corresponding to the pGla promoter and T.T. region was isolated and the DNA was purified

using QiexII kit (Qiagen Inc., Chatsworth, CA, USA). In parallel, the pUC18 plasmid was

digested with PvuII (Promega) and Sspl (Pharmacia Biotech) restriction enzymes, treated

with calf intestinal phosphatase (Pharmacia) and electrophoresed on 1% agarose gel. The 1.8

kb band containing the pUC18 backbone was purified using the QiaxII kit. The two purified

DNA fragments were then ligated together with T4 DNA ligase (Promega) to form a new

expression vector, pGla(NV). The identity of the vector was confirmed by restriction

analysis.

Construction of vector driving expression of A.niger β-galactosidase cDNA and structural gene in fungal cells

A pSP72 plasmid (pSPcDNAN) containing part of the pGla promoter and

β-galactosidase cDNA or structural gene was restricted with Xhol which excises the A. niger

β-galactosidase cDNA and part of the glucoamylase promoter. This DNA fragment was

isolated as an Xhol fragment, purified using QiexII kit (Qiagen) and cloned into pGla(NV)

(Figure 1) predigested with Sail and Xhol. This resulted in reconstructing the full length glucoamylase promoter driving expression of the β-galactosidase cDNA (Fig. 2, step 3).

Restriction analysis and sequencing data confirmed that the resultant construct has a correct

open reading frame, full length pGla promoter and the T.T. region. The cDNA and genomic

expression systems were denoted PglaNVBC (Figure 2) and PglaNVBG, respectively.

Construction of prolactase with bovine lactoferrin used as a tail fused through a pepsin recognition site at the 3'-end of the A. niger β-galactosidase gene (Figure 3. design 1

The bovine lactoferrin (BLF) cDNA was cloned from mRNA isolated from bovine

mammary gland or MAC-T cell line by RT-PCR amplification. Primers were designed

according to Tsang et al. (1991): (5'-GGTCACCGAGCACTGGATGGG-3', from 58 to 78)

and (5'-ATCCGACTTCAGGAAGGGAGA-3', from 2351 to 2371 numbering according to

the published sequence). The PCR was performed in a final volume of 100 μl reaction using

the ExpandLong system (Stratagene, La Jo 11a, CA, USA) according to the manufacture's

protocol. The reaction was carried out using MiniCycler (MJ Research Inc., Watertown, MA,

USA) for 35 cycles. Each cycle consisted of 30 sec. at 94°C, 80 sec. at 60 °C and 2 min. at

72 °C. The sequence coding for the mature BLF was amplified using primers (5'-

CTGTCAGTCAGTTAGACGTCGAAAGCGGCCGCTT

ACCTCGTCAGGAAGGCGCAGGCTT-3') and (5'-CTTTGGTACCGTTTCTGCAA

GGCCCCGAGGAAAAACGTTCGA-3'). The 3' sequence of the β-gal genomic DNA was

also amplified by (5-ACCCTGTGGGCTCTCGACTCT-3') and

(5'-CTTGCAGAAACGGTACCAAAGGTATGCACCCTTCCGCTTCTTGTACTTGG-3').

The underlined sequences represent overlapping sequences so that the amplified 3' sequence

of the β-gal genomic DNA was fused with the BLF sequence using PCR without primers.

The PCR conditions and cycling profile were the same as shown above except for the

annealing temperature that was performed at 50 degrees C. The fusion product was then subcloned into a pGEMT vector that allows direct subcloning of PCR products. The resulting

vector pGEMT/bgblfPCR was restricted with Nael and SpHI and ligated into the Nael, SpHI

restricted pGlaNVBC expression vectors (Figure 2, step 3). The resulting vector was

designated CTF8 (Figure 3, step 3) and allows expression (under the control of the pGla

promoter) of the β-galactosidase cDNA fused to bovine BLF through a pepsin site. This site

was generated by the sequences at the overlapping region (underlined) of the primers above

and encodes for LWYRFCK. The 3 '-primer used to amplify the BLF contained stop codons

in all three possible reading frames, a Notl and a Aatll site. A similar approach was used to

construct the expression system that carries the genomic β-galactosidase gene (GTF7).

EXAMPLE 2

Construction of prolactase carrying poly amino acid tails fused at the 3'-end of the β- galactosidase gene through a pepsin recognition site

The basic design in this series of constructs was to have polyamino acid tails of two

lengths (for example, 16 and 25 amino acids) that possess either positive (Arg tail) or

negative (Asp tail) charge. These tails are to be fused to the 3'-end of the β-galactosidase

gene through a pepsin recognition site. Since in this series of experiments we utilized fungal

expression systems as the host organism which are known to secrete pepsin-like enzymes it

was reasoned that the pepsin sites we constructed in may be cleaved by these pepsin-like

molecules. We therefore constructed expression vectors for each polyamino acid tail with or

without the pepsin site (see Table 2 for details). A total of eight expression constructs

carrying poly-amino-acid tails at the 3'-end of the β-galactosidase genomic gene (Table 2)

were constructed using as a backbone the vector pGgbpb (Figure 4, step 1). Four linkers

encoding for the desired number of poly-amino-acids were first synthesized (Immunocorp, Montreal, Quebec, Canada). Each linker contained an Agel and a Notl overhang and an

internal BSSHII site (Figure 4, step 2). For the pepsin minus constructs, the linkers were

cloned directly into the pGgbpb vector between Agel and Notl sites (Figure 4, step 3). For the

constructs with the pepsin cleavage site, the linkers were digested with BSSHII restriction

enzyme (New England Biolabs Ltd., Mississauga, ON, Canada), dephosphorylated using

alkaline phosphatase (Pharmacia Biotech) and cloned into the pGgbpb vector between the

BSSHII and Notl sites (Figure 4, step 4). The integrity and open reading frame of each

construct was verified by DNA sequencing analysis.

TABLE 2

EXAMPLE 3

Construction of prolactase containing bovine -lactalbumin (used as the regulatory domain fused at the 3'-end of the β-galactosidase gene with or without a pepsin recognition site

Bovine α-lactalbumin was cloned by RT-PCR. Total RNA was first extracted from

mammary gland samples of a lactating cow using Tri-Zol^Tm (GibcoBRL, Burlington, ON,

Canada) and then reverse-transcribed into first strand cDNA using the First Strand cDNA

Synthesis kit (Pharmacia). Two primers (5-TGCGGAATTCGAGCAGTGTGG TGACCCCATTT-3'; 5'-TAATGCGGCCGCAGCAAAGACAGCAGGTGTTC-3') were

designed based on the sequence of bovine α-lactoalbumin cDNA (α-lac) (Vilotte et al., 1987)

to amplify the α-lac cDNA (Figure 5, step 1). The PCR was carried out in a final volume of

100 μl reaction using the Taq DNA polymerase (Boehringer Mannheim Canada, Laval, QC,

Canada) according to the manufacture's protocol. The reaction was carried out using a

MiniCycler (MJ Research Inc., Watertown, MA, USA) for 35 cycles. Each cycle consisted of

30 sec. at 94°C, 80 sec. at 60 °C and 2 min. at 72 °C. The PCR product was subjected to

restriction analysis and cloned into pCDNA3 (Clontech) vector between EcoRI and Notl

sites. The identity of the PCR product was confirmed by DNA sequencing analysis.

To construct the mature α-lac as a tail, primers

(5'-AGCTGCGCGCTACCGGTCAGAACAGTTAACAAAATGTGAGGTGTTCC-3' and

5'-TAATGCGGCCGCAGCAAAGACAGCAGGTGTTC-3') were used using as templates

for the cloning of α-lac into the pCDNA3. The PCR product was subcloned into the pGgbpb

vector

(Figure 4 & 6) between BssHII and Notl resulting to the fusion of a-lac as a tail at the 3-end

of

β-galactosidase through a pepsin recognition site (Figure 5, step 2). Alternatively, the a-lac

PCR fragment was digested with Agel and Notl and ligated to the Agel, Notl sites of the

pGgbpb vector (Figure 5, step 3). The resulting plasmid contains the a-lac gene fused at the

3'-end of the beta-gal gene without the pepsin site. The open reading frame of each construct

was verified by DNA sequencing analysis. EXAMPLE 4

Construction of prolactase carrying β-galactosidase cDNA fused to the β-galactosidase through a pepsin site (as the regulatory domain)

Strategy of construction

The objective was to construct an expression cassette, pGgbpb (Fig. 6), based on the

promoter region of the Aspergillus niger glucoamylase gene and the following DNA

elements: the glucoamylase promoter (Element 1), full length genomic β-galactosidase gene

sequence (Element 2), β-galactosidase cDNA used as tail/regulatory domain (Element 4),

and the glucoamylase polyA site (Element 5). A short sequence (Element 3) encoding a

recognition site for pepsin was designed to fuse the two β-galactosidase genes (Element 2 and

Element 4).

Vector

The starting vector is pGTF7 (Fig. 9). This vector contains pUC18 backbone, Element

1, Element 2 and Element 5. There is a Notl site between Element 2 and Element 5, which

will be used for subcloning.The other vectors used in this construction are pSPganb3' (Fig. 7)

and pSPanb (Fig. 8) which are pSP72 vectors containing genomic and cDNA β-galactosidase

gene sequences, respectively.

Strategy

The construction replaced Element 2 of the plasmid pGTF7 (Fig. 9) with a DNA

fragment containing Element 2, Element 3 and Element 4 in the three following steps:

1. Modification of the 3' end of genomic β-galactosidase gene (Element 2) by PCR (Fig. 7).

The DNA template is the plasmid pSPganb, which contains the A. niger β-galactosidase

genomic clone. The 5' primer was 5'-CAAGAACGGCATCTGGTCAG-3', and the 3' primer

is 5'-ATTACTGCAGCGCGCCAGCAGAGCGTAGAAGGACCGGTATGC

ACCCTTCCGCTTCT-3'. The PCR amplification has performed in a 100 μl reaction

containing 5 ng of pSPganb, 2.5 u of PWO DNA polymerase (Boehringer Mannhein), 0.2

mM of each deoxyribonucleotide(dNTP), 0.25 uM of each primer and the thermo DNA

polymerase buffer. The reaction was carried out in MiniCycler (MJ Research Inc.) using the

following program: denaturation at 94°C for 3 min; 30 cycles each consisting of 1 min at

94°C, 1 min at 60°C, 2 min at 72°C; and a final extension step of 7 min at 72°C. The PCR

product was identified as a lkb band on 0.8% agarose gel, purified using QIAEX Gel

Extraction Kit (QIAGEN Inc, 20020), digested with SacII and Pstl, purified as a 0.7 kb DNA

fragment from an agarose gel using QIAEX Gel Extraction Kit, ligated to SacII, Pstl

restricted pSPganb plasmid (5. lkb DNA fragment) resulting to plasmid pSPganb3' (Fig. 7).

The pepsin recognition site (Element 3) and suitable cloning sites are (Agel and BssHII)

generated in the PCR product.

2. Modification of the 5' and 3' ends of cDNA β-galactosidase gene (Element 4) by

PCR. Suitable cloning sites are designed to fuse the β-galactosidase cDNA sequences at the

3'-end of the genomic β-galactosidase gene. The DNA template for PCR was plasmid pSPanb

(Figure 8). The 5' primer is 5'-ATTAGCGCGCGAACTGTTGCAGAAATACGTC-3*, and

the 3' primer is

5'-ATTACTCGAGCTAGTCAGTTAGACGTCGAAAGCGGCCGCTAGTATGCA

CCCTTCCGCTT-3'. PCR amplification was performed in a lOOμl reaction containing 5 ng

of pSPanb, 2.5 u of PWO DNA polymerase (Boehringer Mannhein), 0.2 mM of each

deoxyribonucleotide(dNTP), 0.25 uM of each primer and the thermo DNA polymerase buffer. The reaction was carried out in MiniCycler (MJ Research Inc.) using the following

program: denaturation at 94°C for 3 min; 30 cycles each consisting of 1 min at 94°C, 1 min at

60°C, 4 min at 72°C; and a final extension step of 7 min at 72°C. The PCR product was

identified on 0.8% agarose gel as a 2.9kb band, purified using QIAEX Gel Extraction Kits,

digested with BssHII and Xhol, and it was subcloned into pSPganb3' (Fig. 7) to generate

plasmid pSPgbpb (Fig.8) which contains Element 2, Element 3 and Element 4.

3. Construction of pGgbpb.

The Xbal/Notl DNA fragment from pSPgbpb (Fig. 8) containing Element 2, Element

3 and Element 4 is cloned into pGTF7 to generate the expression cassette pGgbpb (Fig. 9).

EXAMPLE 5

Construction of a prolactase vector containing bovine k-casein as the regulatory domain of activity of β-galactosidase

1. Cloning of the K-casein gene (cDNA)

RNA was extracted from bovine mammary gland tissue. Using the Pharmacia T-

primed First Strand Kit (Ready-To-Go, cat. No. 27-9263-01) cDNA was synthesized.The

cDNA served as a template for the cloning of the κ-casein gene.

Primers were generated spanning the entire gene (.750kb) which contained unique

restriction sites (EcoRI, BamHI). PCR was performed using the PWO enzyme. After

amplification a 0.75kb product was generated, which was restricted with EcoRI and BamHI

and subcloned into the pUC18 vector (see Figure 10, step b).

2. Cloning the κ-casein gene into an A.niger expression vector (pGLA)

In order to subclone the K-casein gene into the pGLA vector only the sequence encoding the mature protein was amplified. For this reason two new primers were generated:

one which primes at the 5' end contains a unique Kpnl site and encodes the first amino acid of

the mature protein. The second 3' end primer contains a unique Not I site to facilitate

subcloning into the pGLA vector.

The PCR product (.56kb) was restricted with bpnl/Notl (step 2) and subcloned into

the pGLA vector, restriced with Kpn I and Not I.

Hydrolysis of Lactose by Prolactase Activation under Simulated Gastric Juice

Conditions

Objective

In order to determine if latent lactase activity can be recovered from the purified

prolactase enzyme (reversibly inactivated) the following experiments are performed. The

process is that pepsin will be cleaving the pepsin recognition site (between the b-gal gene and

the tail / regulatory domain) thereof releasing the lactase counterpart of the prolactase

molecule allowing it to act / hydrolyze the lactose. Lactose hydrolysis is measured by a

variety of methods including HPLC analysis and synthetic substrates (ONPG etc.).

General

The model system developed is based on conditions prevailing in the stomach. Gastric

juice is a simple fluid containing 150 meq/ml of hydrogen (pH of 1.5) and 0.5 to 1 mg/ml of

pepsin. The pure juice at high rates of secretion can have a pH below 1 , but upon

consumption of a meal the pH of the gastric chyme can increase to a pH of 6. Fasting gastric

pH has been estimated to range between 1.5 and 2.5. A range of pepsin concentration from 0.1 to 10 mg/ml is used in order to take into account the great variability of pepsin found in

the human population. Since human pepsin is unavailble commercially the simulated gastric

juice will employ porcine pepsin from Sigma Chemical Co., St Louis, Missouri or the

equivalent.

The activity of the prolactase is tested using 400 ul of milk by the addition 3 different

amounts of hydrochloric acid, pepsin and prolactase or lactase (used as the positive control).

Varying the quantity of hydrochloric acid added allow us to cover a range of pH from about

3.5 to the pH of milk thus around 6.5 to 6.6. Reactions are carried out at 37°C for 2 hours.

Lactose concentrations are determined by HPLC analysis.

Procedure

1). Stock solutions: 150 meq H /L solution pH 1.5, 0.005 M acetate solution pH 4.3,

pepsin stock solution (100 mg/ml).

2). Hydrolysis reactions : Pipet 400 ul of milk, add 0 or 200 or 600 ul of hydrochloric

acid, add the appropriate volume of pepsin solution (range 0.1 - 10 mg/ml), adjust the volume

with deionized water, add the lactase or prolactase, place the tubes in a water bath at 37 °C

and incubate for 2h. The reactions are terminated by boiling for 10 min and stored at -20 C

until analyzed by HPLC.

3). Lactose analysis

Triplicate samples of each reaction are analyzed for lactose concentration by HPLC.

4). Calculations

For each set of conditions controls with no lactase or prolactase were done allowing the percentage of lactose hydrolysis due to the enzyme addition to be calculated.

% hydrolysis = (% lactose in the "hydrolyzed" sample/% lactose in the control) *100%

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What is claimed is:

Claims

Claims:

1. A synthetic ╬▓-galactosidase enzyme which differs from naturally-occurring

╬▓-galactosidase in that said synthetic enzyme is substantially inactive when present in milk,

and is activated by a chemical or condition naturally present in the gastrointestinal tract of

humans.

2. The synthetic enzyme of claim 1, wherein said enzyme comprises a peptide or

polypeptide linked to ╬▓-galactosidase via a cleavage site therebetween which is cleavable by

an enzyme which is present in the gastrointestinal tract of humans and substantially absent

from milk, wherein said cleavage site is substantially uncleavable by any chemicals or

conditions of milk.

3. The synthetic enzyme of claim 2, wherein said cleavage site is a peptide bond

cleavable by an enzyme in the human gastrointestinal tract.

4. The synthetic enzyme of claim 1, wherein said ╬▓-galactosidase is of A. niger

origin.

5. The synthetic enzyme of claim 1, comprising a signal peptide which causes

secretion of the enzyme from cells in which it is expressed.

6. A DNA molecule encoding the enzyme of claim 3.

7. A recombinant cell comprising the DNA molecule of claim 6.

8. A transgenic mammal whose mammary glands express the DNA molecule of

claim 6, wherein said DNA molecule includes a mammary-specific promoter controlling

transcription of the DNA encoding said synthetic enzyme, and further includes a nucleic acid

sequence encoding a signal peptide which causes secretion of said enzyme into the milk of

said transgenic mammal.

9. The synthetic enzyme of claim 2, wherein said cleavage site is a peptide bond

cleavable by an enzyme or condition used in cheese processing.

10. The synthetic enzyme of claim 9, wherein said enzyme used in cheese processing

is chymosin.