Journal of Pharmaceutical & Biomedical Analysis
`Vol . 7, No . 2, pp . 173-183 .1989
`Printed in Great Britain
`
`0731-7085i89 $3 .00 + 0 .00
`©1989 Pergamon Press plc
`
`Characterisation of a secreted form of
`recombinant derived human growth hormone,
`expressed in Escherichia coli cells*
`
`PAR GELLERFORS,t GUNILLA EKETORP, KARIN FHOLENHAG, BOHDAN PAVLU,
`STIG JOHANSSON and LINDA FRYKLUND
`
`KabiVitrum Peptide Hormones AB, S-112 87 Stockholm, Sweden
`
`Abstract : Recombinant DNA derived human growth hormone (rhGH), Genotropin ® ,
`has been expressed in E. coli cells as a pre-hormone, where the heat stable enterotoxin II
`signal peptide (STII) was linked to hGH to get secretion of the hormone to the
`periplasmatic space . The pre-hormone was efficiently cleaved during secretion, by an
`endogenous signal peptidase generating the correct N-terminal (Phe) end as shown by
`protein sequence analysis . The purity of rhGH was studied by SDS-PAGE, in
`combination with laser densitometry and HI-HPLC . These techniques showed that the
`level of modified rhGH forms, e .g . aggregated and proteolytically cleaved (16 and
`6 kDa) in the preparation was in the 0 .5-1% range .
`Furthermore, evidence that the correct disulphide bonds (Cys53-CYs1a5 ; CYs182 - Cysls9)
`were formed in rhGH during secretion has been shown by a combination of tryptic
`fingerprint and amino acid analysis . CD-spectroscopic analysis suggested an identical
`secondary structure to that of pituitary derived human growth hormone (pit-hGH) .
`Isoelectric focusing revealed an isoelectric point (p1) for rhGH of 5 .0 similar to pit-hGH
`and in excellent agreement with the theoretical value 5 .1, based on the primary
`sequence . Finally, an apparent molecular weight of 22,000 was obtained for rhGH, by
`SDS-PAGE . All these physico-chemical studies suggest that rhGH is structurally
`identical to pit-hGH, somatotropin .
`
`Keywords: Human growth hormone, recombinant DNA, secretion, E . coli .
`
`Introduction
`
`A major task in modern biotechnology has been to establish the safety and efficacy of
`recombinant DNA-derived protein pharmaceuticals . One obvious advantage of re-
`combinant DNA produced products is the possibility of well controlled production
`
`' Presented at the Symposium on "Biomolecules - Analytical Options", May 1988, Sollentuna, Sweden .
`tTo whom correspondence should be addressed .
`Abbreviations : SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis ; kDa, kilodalton ;
`ELISA, enzyme-linked immunosorbent assay ; DTE, dithioerythritol ; RP-HPLC, reverse phase high-
`performance liquid chromatography ; HI-HPLC, hydrophobic interaction high-performance liquid chroma-
`tography ; CD, circular dichroism ; pit-hGH, pituitary derived human growth hormone ; STII-rhGH, heat stable
`enterotoxin II signal peptide (23 amino acid residues) linked to rhGH (191 amino acid residues) .
`
`173
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`MYLAN INST. EXHIBIT 1100 PAGE 1
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`MYLAN INST. EXHIBIT 1100 PAGE 1
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`

`174
`
`PAR GELLERFORS et al .
`
`systems . However, by heterologous expression in E. coli or yeast for example, several
`questions have been raised concerning : (i) the possibility of wrongly folded proteins ; (ii)
`the presence of secreted precursor forms of signal peptide-containing proteins ; and (iii)
`proteins having post-translational modifications caused by deamidation, proteolytic
`cleavage etc .
`Human growth hormone (met-hGH) has been synthesised in E. coil, as a cytoplasmic
`protein with an extra methionine residue in the N-terminal end [1] . However, recent
`advances in the understanding of the underlying mechanisms of protein secretion in E.
`coli, has made it possible to make a secreted form of the hormone . This paper describes
`the synthesis and secretion of authentic human growth hormone (rhGH), Genotropin ® ,
`in E. coil . The secretion, which mimics the natural process of the somatotropic cells in
`the pituitary gland, was achieved by linking the gene for the heat-stable enterotoxin II
`(ST11) signal peptide (23 amino acids) [2], to the hGH gene (191 amino acids) . By this
`construction rhGH is secreted to the periplasmic space [3], from where it could be easily
`purified to homogeneity . The analytical techniques which have been used to characterise
`the rhGH preparation with respect to structure and purity are presented here .
`
`Materials and Methods
`
`Protein sequence analysis was carried out on an Applied Biosystems Inc . gas phase
`protein sequencer 470A (Applied Biosystems, Foster City, CA, USA) .
`SDS-PAGE was carried out essentially as described by Laemmli [4] . Quantitation of
`silver-stained SDS-PAGE gels was done by laser densitometry (633 nm) using an LKB
`UltroScan XL laser densitometer (LKB, Bromma, Sweden) [5] . The hydrophilicity plot
`of rhGH based on the primary sequence was obtained using a computer program "DNA
`Inspector II" (Textco, New Hampshire, USA) .
`H1-HPLC was carried out on a Hewlett-Packard HP 1090 chromatographic system
`(Waldbronn, FRG) using a TSK-Phenyl 5PW column (75 x 7 .5 mm ; Tosoh, Japan [6]) .
`Circular dichroism spectra were recorded on a JASCO J-41A spectropolarimeter
`(Japan Spectroscopic Co ., Tokyo) . The analysis was carried out in 1 mM sodium
`phosphate, 0 .15 M NaCl, pH 6 .8, at a protein concentration of 1 .5 mg ml - ' . The spectra
`were recorded at room temperature .
`Tryptic fingerprint analysis was carried out essentially as follows : rhGH was dissolved
`in 0 .1% bicarbonate buffer (0 .7 mg ml - ') and incubated with TPCK-treated trypsin
`(5 µg ml- ') for 2 h at 37°C . Tryptic fragments were subsequently separated by RP-
`HPLC on a Vydac 218 TP C1 ,-column, using an acetonitrile (5-60%) gradient in 0 .1 M
`phosphate buffer (pH = 2 .0) at a flow rate of I ml min -1 .
`Amino acid determination was carried out on an automated amino acid analyser (LKB
`4150 Alpha Plus, LKB-products AB, Sweden), essentially according to the procedure
`described by the manufacturer . The isoelectric point (pI) of rhGH was determined by
`isoelectric focusing in LKB Agarose Z using Pharmalyte, pH 4-6 .5 . The determination
`of the pH-gradient obtained after isoelectric focusing was achieved by a pH surface
`electrode (Ingold 430-3-M8) . A plot of pH versus distance from cathode was
`constructed . The linear part (regression coefficient = 0 .997) was chosen for the pl
`determination .
`Recombinant derived human growth hormone, rhGH, Genotropin ® , used in this study
`was obtained from KabiVitrum Peptide Hormones AB, Stockholm, Sweden . All
`reagents used were of highest available purity .
`
`MYLAN INST. EXHIBIT 1100 PAGE 2
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`MYLAN INST. EXHIBIT 1100 PAGE 2
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`
`

`

`CHARACTERISATION OF A SECRETED FORM OF rhGH
`
`175
`
`Results
`
`Signal peptide processing
`The effective cleavage of the STII signal peptide is carried out by an endogeneous E.
`coli signal peptidase, creating the correct N-terminal end (Phe) of human growth
`hormone (Fig . 1) . Automated Edman degradation analysis of 15 steps (cf. Table 1)
`completely matched the known sequence of human growth hormone [7] . No alanine or
`tyrosine residues belonging to the STII signal peptide were detected in the first
`degradation cycle, showing that the cleavage is highly specific . The detection level for
`underlying secondary sequences is usually in the 2-5% range .
`
`Purity of rhGH
`Figure 2 shows the purity of rhGH following each individual purification step as
`deduced by SDS-PAGE and silver staining . The 22 kDa molecular weight form of rhGH,
`was identified in the periplasmic extract (lane 1) by Western blot analysis (not shown) .
`This form was present together with a few other [25-30] E. coli polypeptides . Also
`present in the periplasmic extract was a proteolytically cleaved form of rhGH with an
`apparent molecular weight of 24 kDa, which upon reduction was split into a 16 and 6 kDa
`form . The 24 kDa form has been fully characterised and shown to have been cleaved in
`the polypeptide chain after Tyr142 . The 24 kDa form, which is fully biologically active in
`the hypophysectomised rat weight-gain assay, was removed during purification down to
`
`met -lys-lys-asn-ile-ala-phe-leu-leu-
`-23
`-15
`
`ala-ser-met-phe-val-phe-ser-ile-ala-
`-10
`
`thr-asn-ala-tyr-ala - PHE-PRO-THR-
`-g
`-~
`
`enteratoxIn signal . •
`sequence
`
`- hGH
`
`signal peptidase
`
`Figure 1
`Escherichia coli signal peptidase cleavage of rhGH
`prehormone (STH-rhGH) .
`
`Table 1
`Sequence determination of rhGH, Genotropin ®
`
`Step
`
`Amino acid identified
`
`Amount (nmol)
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`
`Phe
`Pro
`Thr
`lle
`Pro
`Leu
`Set
`Arg
`Leu
`Phe
`Asp
`Asn
`Ala
`Met
`Leu
`
`31 .5
`17 .5
`10 .6
`16 .3
`10 .4
`12 .9
`4 .4
`7 .5
`6 .9
`7 .1
`4 .5
`5 .6
`5 .8
`9 .3
`7 .4
`
`MYLAN INST. EXHIBIT 1100 PAGE 3
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`MYLAN INST. EXHIBIT 1100 PAGE 3
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`

`

`176
`
`PAR GELLERFORS etal .
`
`Figure 2
`SDS-PAGE of fractions from the purification process . Ten micrograms of protein was loaded on each lane .
`The sample in lane 6 has been lyophilised prior to electrophoresis . Molecular weight assignments were made
`relative to molecular weight markers (cf . Materials and Methods) .
`
`the 0.1-0 .5% level . Furthermore, an aggregated form (molecular weight 44 kDa)
`appeared after freeze-drying of rhGH (lane 6) . The content of this form was dependent
`on the formulation buffer as well as the freeze-drying parameters chosen . The amount of
`the aggregated form was usually in the 1-2% range . No aggregated form was found
`earlier in the purification process . Lanes 2-6 show the removal of contaminating
`polypeptides during the purification process . The level of E. coif proteins after the final
`purification step was extremely low, usually in the 1 ppm (0 .0001%) range and thus
`could not be quantitated by SDS-PAGE and silver-staining . Accurate quantitation was
`achieved by an ELISA-method (unpublished) .
`To more accurately quantitate various molecular weight forms of rhGH present in the
`final preparation, a laser densitometric method in combination with SDS-PAGE was
`developed . Figure 3 shows a laser densitometric determination of aggregated rhGH as
`well as the cleaved (16 and 6 kDa) form of the hormone . The levels of these forms were
`found to be 2 .2 and 0 .5%, respectively .
`
`Physico-chemical properties
`Hydrophobic surface . Every protein has a unique distribution ("finger print") of
`hydrophobic and hydrophilic amino acid residues . Figure 4 shows a hydrophilicity plot of
`STII-rhGH, based on its primary sequence . Seven major and three minor hydrophilic
`regions were identified within rhGH . The location (surface versus interior) of these
`regions affects the hydrophilicity-hydrophobicity balance on the surface . This property
`can be exploited chromatographically using HI-HPLC to separate proteins and
`demonstrate identity .
`The rhGH was chromatographed on a TSK-Phenyl column (Fig . 5) . The resolution
`of the method was found to be very high since methionyl-human growth hormone,
`Somatonorm® , (tR = 17 min) could be separated from rhGH, Genotropin ® ,
`(t R = 14
`min), differing only in one extra methionine residue in the N-terminal end (Fig . 5A) .
`
`MYLAN INST. EXHIBIT 1100 PAGE 4
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`MYLAN INST. EXHIBIT 1100 PAGE 4
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`

`

`CHARACTERISATION OF A SECRETED FORM OF rhGH
`
`177
`
`22 KD
`
`1 .741
`
`0c0 5
`
`ee
`
`Aggregates
`
`16 KD
`
`~
`
`'
`
`A
`
`6 KD
`
`9.127
`
`39 .09
`
`6slance (mm)
`
`149 .96
`
`Figure 3
`Laser densitometric determination of cleaved (16 and 6 kDa) and aggregated forms of rhGH . 10 and 0 .25 pig of
`rhGH was electrophoresed in each lane . Each lane was densitometrically scanned (633 nm) after staining . The
`total integrated peak area was determined and used for the estimation of the relative content of each
`component .
`
`Figure 4
`Hydrophilicity plot of STII-rhGH .
`
`0 .
`
`!4
`
`0'5 ' 0'6
`
`017
`
`0 ' 0 0
`
`I10
`
`fraction of length
`
`Furthermore, rhGH elution time was identical to that for pit-hGH, indicating identical
`hydrophilic surfaces (Fig . 5B) .
`Figure 5C shows an expanded HI-HPLC chromatogram of rhGH to clearly identify
`minor peaks . Four extra peaks beside the main rhGH peak could be identified . These
`variant forms have been characterised and are present in low amounts (less than 1%) in
`purified rhGH .
`
`Circular diehroism spectroscopy
`The secondary structure of rhGH was studied by CD-spectroscopy . A CD spectrum
`was recorded between 250 and 320 nm and found to be identical to that for pituitary
`
`MYLAN INST. EXHIBIT 1100 PAGE 5
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`MYLAN INST. EXHIBIT 1100 PAGE 5
`
`
`
`
`
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`

`

`178
`
`PAR GELLERFORS et al .
`
`A.
`
`B.
`
`G .
`
`go-
`so-
`
`Go-
`
`50-
`
`40
`
`30
`
`20
`
`10
`
`220-
`
`300-
`
`Bo-
`
`50 ,
`
`20^
`
`50-
`
`40
`
`30
`
`20
`
`I0
`
`0
`
`0
`
`Time f
`
`20
`
`Time (mm
`
`20
`.)
`
`Time (min .
`
`Figure 5
`Hydrophobic interaction chromatography (HI-HPLC) of somatotropin preparations . A. rhGH and methionyl-
`hGH (1 :1) B . rhGH plus pituitary-hGH (1 :1) ; C. rhGH (expanded 3 .4-fold) .
`
`MYLAN INST. EXHIBIT 1100 PAGE 6
`
`MYLAN INST. EXHIBIT 1100 PAGE 6
`
`
`

`

`CHARACTERISATION OF A SECRETED FORM OF rhGH
`
`179
`
`A
`
`O
`
`pZ
`
`V-
`
`260
`
`270
`
`290
`280
`WAVELENGTH
`
`300
`
`310
`
`320
`
`E 0
`
`-2-
`
`-4-
`
`-
`
`6
`
`_
`
`-10 -
`
`0
`
`-12 -
`
`250
`
`L•
`
`1 •
`
`•
`
`1 C
`
`1 T
`
`Y
`
`200
`
`0
`
`205
`
`1
`
`210
`
`1
`
`215
`
`t
`
`220
`
`1
`
`225
`
`4
`
`230
`
`235
`
`1
`
`240
`
`1
`
`o,o-o-o-o-o .o-o-o-o-o-o.o-o'e
`
`0
`
`,,
`
`~
`
`WAVELENGTH
`
`B
`
`M E
`
` E
`
`-5
`
`A L
`N L
`1
`N P
`E T -10
`S 1
`I C
`D 1
`G T-15
`E V
`
`-20
`
`Figure 6
`CD spectrum of rhGH . Panel A . rhGH (Q) and pit-hGH (s) ; panel B . Far-ultraviolet circular dichroism
`d-,
`spectra of rhGH . Molar ellipticity (0) is expressed as (deg . cm2
`mol) .
`
`derived human growth hormone (Fig . 6A) . Both preparations exhibited a strong
`maximum at 292 nm indicative of tryptophan transitions . The CD spectrum in the far-
`ultraviolet region (Fig . 6B), was identical to that reported for pituitary derived hGH [8]
`as well as for another recombinant DNA derived hGH preparation [9] . This showed the
`characteristic high content of a-helix, with a minimum at 206 nm and a shoulder at
`223 run, in agreement with recent X-ray crystallographic data [10] .
`
`Disulphide bonds
`Two disulphide bonds viz ., Cys53-CY5165 and Cys 182-Cys 189 , have been identified in
`pituitary human growth hormone [7] . The correct disulphide bond pairing in rhGH has
`been studied by a combination of tryptic fingerprint and amino acid analysis . Figure 7
`shows the hGH amino acid sequence with tryptic cleavage sites indicated . Cleavage of
`
`MYLAN INST. EXHIBIT 1100 PAGE 7
`
`MYLAN INST. EXHIBIT 1100 PAGE 7
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`1 80
`
`PAR GELLERFORS et al .
`
`The amino acid sequence of recombinant human somatotropin
`with tryptic cleavage sites indicated
`
`Flgure 7
`Primary sequence, disulphide bonds and tryptic cleavage sites in human growth hormone .
`
`MYLAN INST. EXHIBIT 1100 PAGE 8
`
`MYLAN INST. EXHIBIT 1100 PAGE 8
`
`

`

`CHARACTERISATION OF A SECRETED FORM OF rhGH
`
`181
`
`T2 .T17
`
`T1
`
`Ts
`
`Tta
`
`T7.
`T14
`
`T12
`
`I
`o
`
`I
`10
`
`f
`20
`
`T10
`
`T11
`
`I
`30
`
`Time
`
`I
`n0
`
`I
`w
`
`I
`60
`
`Mb .
`
`Figure 8
`Tryptic fingerprint of rhGH . rhGH was cleaved with TPCK-treated trypsin . Tryptic fragments literated were
`subsequently separated by RP-HPLC on a Vydac C18-column. Tryptic fragment assignments are based on
`amino acid composition determinations . Tryptic fragment Ti s „9 and T6 , 1 , are linked by disulphide bonds .
`
`Table 2
`Amino acid composition for reduced tryptic S-S fragments
`
`Amino acid
`
`Sample T18
`(179-183)
`
`Sample T 19
`(184-191)
`
`Sample T6
`(42-64)
`
`Sample T, 6
`(159-167)
`
`Asx
`Thr
`Set
`Glx
`Pro
`Gly
`Ala
`Cys
`Val
`Met
`lie
`Lea
`Tvr
`The
`His
`Lys
`
`n g
`
`2-0(2)
`1 .0(1)
`
`2.0 (2)
`
`0 .9 (1)
`1-0(1)
`
`0-9(l)
`
`1 .1 (1)
`
`1 .1 (1)
`0 .8(l)
`
`0 .7 (1)
`
`1 .1 (1)
`
`2 .0(2)
`1 .9(2)
`5 .0(5)
`2 .8 (3)
`3 .0(3)
`0 .4
`0 .1
`0 .9(l)
`0 .1
`
`0 .9(1)
`1 .9(2)
`0.9(1)
`1 .8(2)
`
`0 .2
`
`0 .9(1)
`
`1 .1 (1)
`0.1
`0 .4
`0 .1
`
`1 .1 (1)
`
`0 .9(1)
`0 .1
`
`1 .8(2)
`1 .4(2)
`0 .8(l)
`
`0 .1
`
`0 .9 (1)
`
`Values are expressed as nmol peptide .
`Values in parentheses are the theoretical values (Ref. 6) .
`Tryptophan was not determined .
`Asparagine and glutamine are expressed as aspartic acid and glutamic acid, respectively .
`
`MYLAN INST. EXHIBIT 1100 PAGE 9
`
`MYLAN INST. EXHIBIT 1100 PAGE 9
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`

`

`1 8 2
`
`PAR GELLERFORS et al .
`
`rhGH in the non-reduced state (intact disulphide bonds) with trypsin generated 19
`tryptic fragments . All of them, except T3 and T5 which were too small and eluted in the
`solvent front, were separated by RP-HPLC and identified by amino acid analysis (Fig .
`8) . Four of these fragments, e .g . T6-T16 and T18-T19, were linked by disulphide bonds to
`each other and could be isolated . After disulphide bond cleavage (DTE), each individual
`tryptic fragment (T6 , T 16 , T 18 and T19) was again separated by RP-HPLC (not shown),
`and identified by amino acid analysis (Table 2), to establish the correct disulphide pairing
`of rhGH .
`
`1
`
`2
`
`3
`
`IM
`94
`
`67
`
`43
`
`30
`
`20
`
`14.4
`
`Figure 9
`rhGH molecular weight determination by SDS-PAGE . Upper panel . Lane 1, standard proteins with known
`molecular weight ; phosphorylase b (94,000) ; albumin (67,000) ; ovalbumin (43,000) ; carbonic anhydrase
`(30,000) ; trypsin inhibitor (20,100) ; a-lactalbumin (14,400) . Lane 2, rhGH ; lane 3, pit-hGH (Crescormon ® ) .
`Lower panel . A plot of log, 0 molecular weight (MW) versus migration distance .
`
`MYLAN INST. EXHIBIT 1100 PAGE 10
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`MYLAN INST. EXHIBIT 1100 PAGE 10
`
`

`

`CHARACTERISATION OF A SECRETED FORM OF rhGH
`
`Figure 10
`Isoelectric focusing of rhGH . Isoelectric focusing was
`carried out in the pH interval of 4 .0-6 .5 . pH across
`the gel was measured by a surface electrode . Bands
`were subsequently visualised by Coomassie Blue
`staining . The isoelectric point of rhGH was
`determined relative to the measured pH gradient .
`Lane 1, rhGH ; lane 2, pit-hGH .
`
`Table 3
`Properties of rhGH, Genotropin®
`
`Test
`
`Value
`
`183
`
`2
`
`1
`
`Ip 5 .0 -4
`
`Primary structure
`N-terminal amino acid
`Disulphide bonds
`Mw (SDS-PAGE)
`Mw (theoretical)
`Molecular form
`Isoelectric point
`Net charge at pH 7
`
`Single polypeptide chain of 191 amino acid residues
`Phe
`2 (CYs53 -CYsr6s CYslaz-CYsrs9)
`22 .000 ± 500
`22 .096
`Monomer
`5 .0
`-5
`
`Molecular weight and isoelectric point of rhGH. The apparent molecular weight of
`rhGH was determined by SDS-PAGE relative to several standard proteins . A molecular
`weight of approximately 22,000 (log 4 .34) was obtained (Fig . 9), which is in excellent
`agreement with that for pit-hGH (21,500) and the theoretical value (22,098), based on
`the primary sequence for hGH [7] . Furthermore, the isoelectric point of rhGH was
`determined by isoelectric focusing (Fig . 10) . A value of 5 .0 was found, in good
`agreement with pit-hGH . A summary of physico-chemical properties obtained for rhGH
`is given in Table 3 .
`
`Conclusion
`
`The results presented in this paper indicate that rhGH, when expressed and secreted
`from E. coil cells, has all the physico-chemical properties of authentic pituitary derived
`human growth hormone .
`
`References
`
`[1] L . Fryklund, J . Brandt and G. Eketorp et a[., Proceedings of the FDA-USP Workshop on Drug and
`Reference Standards for Insulins, Somatropins, and Thyroid-axis Hormones . Bethesda, Maryland, 19-21
`May, 319-327 (1982) .
`[2] R. N . Picken, A . J . Mazaitis, W . K . Maas, M. Rey and H. Heyneker, Infect. Immun . 42, 269-275 (1983) .
`[3] C . N . Chang, M. Rey, B . Buchner, H . Heyneker and G . Gray, Gene 55, 189-196 (1987) .
`[4] U . K . Laemmli, Nature (Land) 227, 680-685 (1970) .
`[5] S . Johansson, B . Skoog and P . Gellerfors (manuscript in preparation) .
`[6] B . Pavlu, P . Gellerfors and B . Hancock (manuscript in preparation) .
`[7] T . A . Bewley and C . H . Li, Adv. Enzymol. 42, 73-166 (1975) .
`[8] L . A . Holladay, R . G . Hammonds, Jr and D . Puett, Biochemistry 13, 1653-1661 (1974) .
`[9] G . W . Becker and H . M . Hsiung, FEBS Lett. 204, 145-150 (1986) .
`[101 S . S . Abdel-Meguid, H. S . Shieh, W. W . Smith, H. E . Dayringer, B . N. Violand and L . A . Bentle, Proc .
`nain . Acad. Sci . USA 84, 6434-6437 (1987) .
`
`[Received for review 4 May 1988; revised manuscript received 28 June 1988]
`
`MYLAN INST. EXHIBIT 1100 PAGE 11
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`MYLAN INST. EXHIBIT 1100 PAGE 11
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`