Regulation of Casein Kinase-2 (CK2) Activity by Inositol Phosphates

Casein kinase 2 (CK2) was one of the first protein kinases to be discovered and has been suggested to be responsible for as much as one-fifth of the eukaryotic phosphoproteome. Despite being responsible for the phosphorylation of a vast array of proteins central to numerous dynamic cellular processes, the activity of CK2 appears to be unregulated. In the current study, we identified a protein kinase activity in rat liver supernatant that is up-regulated by inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6). The substrate for the inositol phosphate-regulated protein kinase was identified as a phosphatidylcholine transfer protein-like protein. Using the phosphorylation of this substrate in an assay, we purified the inositol phosphate-regulated protein kinase and determined it to be CK2. Bacterially expressed recombinant CK2, however, showed very high basal activity and was only modestly activated by IP6 and not regulated by IP. We found that an endogenous component present in rat 4liver supernatant was able to inhibit both recombinant and liver-purified CK2 basal activity. Under these conditions, recombinant CK2 catalytic activity could be increased substantially by IP4, inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6. We concluded that, contrary to the previously held view, CK2 can exist in a state of low constitutive activity allowing for its regulation by inositol phosphates. The ability of the higher inositol phosphates to directly stimulate CK2 catalytic activity provides the first evidence that these signaling molecules can operate via a direct control of protein phosphorylation. Casein kinase 2 (CK2) was one of the first protein kinases to be discovered and has been suggested to be responsible for as much as one-fifth of the eukaryotic phosphoproteome. Despite being responsible for the phosphorylation of a vast array of proteins central to numerous dynamic cellular processes, the activity of CK2 appears to be unregulated. In the current study, we identified a protein kinase activity in rat liver supernatant that is up-regulated by inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6). The substrate for the inositol phosphate-regulated protein kinase was identified as a phosphatidylcholine transfer protein-like protein. Using the phosphorylation of this substrate in an assay, we purified the inositol phosphate-regulated protein kinase and determined it to be CK2. Bacterially expressed recombinant CK2, however, showed very high basal activity and was only modestly activated by IP6 and not regulated by IP. We found that an endogenous component present in rat 4liver supernatant was able to inhibit both recombinant and liver-purified CK2 basal activity. Under these conditions, recombinant CK2 catalytic activity could be increased substantially by IP4, inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6. We concluded that, contrary to the previously held view, CK2 can exist in a state of low constitutive activity allowing for its regulation by inositol phosphates. The ability of the higher inositol phosphates to directly stimulate CK2 catalytic activity provides the first evidence that these signaling molecules can operate via a direct control of protein phosphorylation. Numerous studies over the last 20 years have established inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP, inositol 4-phosphate; IP2, inositol 1,4 bisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP5, inositol 1,3,4,5,6-pentakisphosphate; IP6, inositol hexakisphosphate; IP7, diphosphoinositide pentakisphosphate; IS6, inositol hexasulfate; CK2, casein kinase-2; rCK2, recombinant CK2; PCTP, phosphatidylcholine transfer protein; DTT, dithiothreitol; cpm, cycles per minute; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LC-MS/MS, liquid chromatography-mass spectrometry/mass spectrometry; RS4, fraction 4 from the resource S column; RS4inac, heat-inactive fraction 4 from the resource S column.1The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP, inositol 4-phosphate; IP2, inositol 1,4 bisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP5, inositol 1,3,4,5,6-pentakisphosphate; IP6, inositol hexakisphosphate; IP7, diphosphoinositide pentakisphosphate; IS6, inositol hexasulfate; CK2, casein kinase-2; rCK2, recombinant CK2; PCTP, phosphatidylcholine transfer protein; DTT, dithiothreitol; cpm, cycles per minute; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; LC-MS/MS, liquid chromatography-mass spectrometry/mass spectrometry; RS4, fraction 4 from the resource S column; RS4inac, heat-inactive fraction 4 from the resource S column. as a classical second messenger molecule (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6157) Google Scholar). However, IP3 is only one of a vast array (numbering potentially as high as 60) of inositol phosphate isomers found within cells (2Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (528) Google Scholar). In contrast to IP3, the biological function of the majority of these inositol phosphates is either unknown or poorly understood. Of particular interest is the phosphorylated product of IP3, namely inositol 1,3,4,5-tetrakisphosphate (IP4), which shows many of the hallmarks of a second messenger molecule. Early studies focused on a role for IP4 in calcium mobilization; however, the cloning of GAP1IP4BP, which shows high affinity binding to IP4 (3Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar), and the recent demonstration that IP4 is involved in chromatin remodeling in yeast (4Shen X. Xiao H. Ranallo R. Wu W.H. Wu C. Science. 2003; 299: 112-114Crossref PubMed Scopus (291) Google Scholar, 5Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (313) Google Scholar) suggest that IP4 function may extend beyond calcium signaling. Of the higher inositol phosphates, possibly the most enigmatic is inositol hexakisphosphate (IP6). Found in all mammalian cells in micromolar concentrations (6Pittet D. Schlegel W. Lew D.P. Monod A. Mayr G.W. J. Biol. Chem. 1989; 264: 18489-18493Abstract Full Text PDF PubMed Google Scholar, 7Szwergold B.S. Graham R.A. Brown T.R. Biochem. Biophys. Res. Commun. 1987; 149: 874-881Crossref PubMed Scopus (111) Google Scholar), the biological function of this molecule has remained elusive. An involvement in endo- and/or exocytosis (8Ohara-Imaizumi M. Fukuda M. Niinobe M. Misonou H. Ikeda K. Murakami T. Kawasaki M. Mikoshiba K. Kumakura K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 287-291Crossref PubMed Scopus (67) Google Scholar, 9Hoy M. Efanov A.M. Bertorello A.M. Zaitsev S.V. Olsen H.L. Bokvist K. Leibiger B. Leibiger I.B. Zwiller J. Berggren P.O. Gromada J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6773-6777Crossref PubMed Scopus (53) Google Scholar) has been indicated by the discovery that a number of proteins involved in this process have a high affinity for IP6 (10Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12Gaidarov I. Krupnick J.G. Falck J.R. Benovic J.L. Keen J.H. EMBO J. 1999; 18: 871-881Crossref PubMed Scopus (166) Google Scholar) and that microinjection of IP6 can regulate synaptic vesicle fusion (13Llinas R. Sugimori M. Lang E.J. Morita M. Fukuda M. Niinobe M. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12990-12993Crossref PubMed Scopus (87) Google Scholar). Recently, the potential biological processes that may be regulated by IP6 have been extended by the demonstration that mRNA export (14York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar), DNA repair (15Hanakahi L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 16Hanakahi L.A. West S.C. EMBO J. 2002; 21: 2038-2044Crossref PubMed Scopus (86) Google Scholar), chromatin remodeling (4Shen X. Xiao H. Ranallo R. Wu W.H. Wu C. Science. 2003; 299: 112-114Crossref PubMed Scopus (291) Google Scholar, 5Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (313) Google Scholar), and protein phosphatase activity (17Larsson O. Barker C.J. Sj-oholm A. Carlqvist H. Michell R.H. Bertorello A. Nilsson T. Honkanen R.E. Mayr G.W. Zwiller J. Berggren P.O. Science. 1997; 278: 471-474Crossref PubMed Scopus (122) Google Scholar) can all be influenced by IP6. In an attempt to address the question of novel signaling properties of the inositol phosphates, we tested the possibility that protein kinases may exist that are regulated directly by inositol phosphates. The rationale was that, of the established second messenger molecules (e.g. cAMP, cGMP, diacylglycerol, and calcium), the inositol phosphates are unique in that they do not appear to directly activate a protein kinase. In our initial studies, we partially purified a protein kinase from porcine brain that was able to phosphorylate the synaptic vesicle-associated protein pacsin/syndapin I in an IP6-dependent manner (18Hilton J.M. Plomann M. Ritter B. Modregger J. Freeman H.N. Falck J.R. Krishna U.M. Tobin A.B. J. Biol. Chem. 2001; 276: 16341-16347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). This was the first demonstration of an inositol phosphate-regulated phosphorylation and supported a role for IP6 in the control of synaptic vesicle endocytosis. Importantly, the kinase that was identified in porcine brain was only activated by IP6 and isomers of IP7. No activated phosphorylation was observed using the lower inositol phosphates, in particular IP3 or IP4 (18Hilton J.M. Plomann M. Ritter B. Modregger J. Freeman H.N. Falck J.R. Krishna U.M. Tobin A.B. J. Biol. Chem. 2001; 276: 16341-16347Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the current study, we have continued with our investigation of inositol phosphate-regulated protein phosphorylation. We report here that phosphatidylcholine transfer protein (PCTP)-like protein, purified from a rat liver supernatant preparation, is a substrate for a protein kinase that is regulated by IP4, IP5, and IP6. Purification of the inositol phosphate-regulated protein kinase revealed the identity of the kinase to be the ubiquitously expressed serine/threonine kinase casein kinase 2 (CK2). Our data demonstrate that CK2, far from being constitutively active as previously thought, is in fact under tight inhibitory control and that, under these conditions, inositol phosphates are able to activate CK2 catalytic activity. Materials—All inositol polyphosphates (with the exception of IP4 and the isomers of IP4) were purchased from Sigma-Aldrich Company Ltd. (Poole, Dorset, UK). d-inositol-1,3,4,5-tetrakisphosphate and the isomers of IP4 were synthesized at the of was purchased from expressed recombinant CK2 was from was from for of from the were in kinase 20 with and in the or of inositol phosphates in a of a at the was by the of proteins were on a which was and for recombinant CK2 was of kinase were with and of S fraction 4 in and in in the or of inositol phosphates 20 of at a of phosphorylation the was and as a preparation, of S fraction 4 were for at on and for at to of the supernatant fraction were used in CK2 of on S rat were in of with and The was at for 20 and the supernatant was at for The supernatant was to a S and with of The was with a of at a of in a of were and for kinase activity. The majority of inositol phosphate-regulated protein kinase activity at and on and 4 from the S inositol phosphate-regulated protein kinase were and to a The was with 2 of and with a of in were and for kinase activity. this the inositol phosphate kinase activity was were kinase the were of and used in the kinase In this it was determined that the inositol phosphate-regulated protein kinase was with and and the substrate protein was with and on using S and were and the kinase and and the substrate were and the The kinase was an and the was with of were from the with the at in 20 phosphate-regulated protein kinase activity was identified in which were by The substrate was on a using the In this the substrate protein was by on from the and the inositol phosphate-regulated protein kinase activity were of with and to a The was with the and first with a of and with in were and for kinase activity. The inositol phosphate-regulated protein kinase activity was from the with and be that the inositol phosphate-regulated activity was and to the kinase with a low basal activity and in a state able to be regulated by inositol phosphates, the to be over a of 2 from using an were with and with in with porcine using an from the with of were by either and/or MALDI-TOF, of the were with and and an of a of in and were on to a using an was on a using a of data were by and protein identity was determined by to the and data The were by using a and a The were a and with for The were the and with a at of in The was at a of and to of the at the of a in was used to for using The were by and protein identity was determined by to the and data were and for an using the protein data of were synthesized as by A.M. Chem. 1997; Scopus Google Scholar). The was by and and was as being and of Bacterially liver was using The was using The was used in with to the and of protein to the protein with and at the and protein was at the to a fusion protein was to the of the protein. protein to was used in kinase of 20 of in kinase and recombinant CK2 in a of The was to for 20 and was by the of were by The were with to the fusion proteins to an was using a with the was using were using the protein were using the of high supernatant from rat liver was over a S and fraction was tested in a protein kinase for the of inositol phosphate-regulated protein phosphorylation the majority of the of inositol phosphates in the not the of phosphorylation However, in an a higher of phosphorylation in the of IP6 and IP4 not revealed that phosphorylation of the protein was by IP4, IP5, and IP6 not the lower inositol phosphates Importantly, in control inositol not phosphorylation of the protein of the from S the inositol phosphate-regulated phosphorylation were and on a column. fraction in a kinase with IP6 revealed for a very in fraction was concluded that this the kinase and which were present in This was tested by in an attempt to the phosphorylation activity. was found that, by fraction with fraction the inositol phosphate-regulated phosphorylation of the protein was data were fraction and fraction not was concluded that the inositol phosphate-regulated kinase and the substrate protein were in and fraction which the kinase and which the fraction was at for This was to the kinase activity. of fraction not the kinase with fraction However, of fraction phosphorylation of the protein with fraction not of fraction not the kinase with fraction of fraction phosphorylation not was concluded from these studies that and from the the substrate for an inositol phosphate-regulated kinase that was present in fraction Purification and of the for the and from the were and on a column. The of the substrate protein in the was determined in kinase the inositol phosphate-regulated kinase was of the protein in and from the This to the of a protein at that with the phosphorylated in the kinase of of the protein in of the which the protein of the identity of the protein substrate was from by of Purification and of the from the which was to kinase activity was on a column. fraction was for inositol phosphate-regulated kinase activity using purified protein as a The inositol phosphate-regulated kinase activity in not were and on a column. The inositol phosphate-regulated kinase activity from this in and The of this kinase activity with the of protein at and of of the to of the that with the of CK2 This identity was by of 2 of identified these protein as a of and of CK2. The of both and was by for not and determined by using an the of CK2 The phosphorylation of protein by CK2 was in studies using recombinant protein expressed as a fusion protein in kinase with expressed recombinant CK2 In these was not phosphorylated by recombinant CK2. In protein was phosphorylated by CK2 in a manner that was to the of IP6 of the of an of the studies, we that the activity of recombinant expressed CK2 be to inositol phosphates. However, using either protein or as a it was found that activity was increased only modestly by IP6 and not at all by IP4 In these constitutive activity was very a with this In the constitutive activity of endogenous CK2 present in the rat supernatant in the first S appears This is on the fact that protein phosphorylation in these is in the of inositol phosphates data suggest the of a of CK2 that is able to CK2 constitutive activity in these rat liver We that, under CK2 constitutive activity was low of the of the inositol phosphates were able to CK2 catalytic possibly by the this we fraction 4 from the S which to CK2 activity in a low basal state This endogenous protein kinase activity and In the of RS4, it was found that the basal activity of was by Under these conditions, the of IP6 was able to activity by data demonstrate that could be regulated in an inositol manner and that this was on low constitutive activity by a component present in a rat liver supernatant was that, the of liver CK2, the ability of the kinase to be regulated by inositol phosphates was of regulation of the liver-purified CK2 was by an in the basal catalytic activity and could be only the process was over a This suggested that the was and This was supported by the that liver-purified CK2, which the ability to be regulated by inositol phosphates, could be to a regulated state by the of This with a in the basal activity observed in the of Using in the of RS4, we were able to the ability of inositol phosphates to stimulate activity. was found that IP4, IP5, and IP6 were able to stimulate phosphorylation of protein and not with IP6 the The lower inositol phosphates which is were to stimulate activity. Importantly, the control was to stimulate activity data are to the data in and evidence to the fact that CK2 is the inositol phosphate-regulated protein kinase responsible for protein phosphorylation in rat liver supernatant Despite CK2 being one of the first protein kinases to be discovered years J. Biol. Chem. Full Text PDF PubMed Google Scholar), the by which this kinase is regulated has remained B. B. S. L.A. 1999; PubMed Scopus (87) Google Scholar, Biochem. J. 2003; PubMed Scopus Google Scholar). In the current study, we have the of an inositol phosphate-regulated protein kinase in a rat liver supernatant Purification of this protein kinase identified it as CK2. We found that the ability of CK2 to be regulated by the inositol phosphates is on the kinase being in a state of low basal activity. This is by a that with CK2. In the of the CK2 basal activity is and under these conditions, inositol phosphates can the catalytic activity of the kinase. studies the first evidence for the of a signaling that CK2 activity. CK2 is a serine/threonine protein kinase that as a of and The is the catalytic and is the product of and The is and is involved in substrate and of the kinase This can exist or or with The of CK2 and its has to the that to one-fifth of the eukaryotic is the of phosphorylation L.A. J. Sci. 2002; PubMed Scopus Google Scholar). of the for CK2 are involved in dynamic cellular as and of L.A. J. Sci. 2002; PubMed Scopus Google Scholar). CK2 a central in the phosphorylation and regulation of proteins involved in dynamic cellular its activity appears to be unregulated. The constitutive activity of CK2 has the that in the phosphorylation of CK2 on regulated L.A. Biophys. PubMed Scopus Google Scholar). Our studies to a protein kinase that be directly regulated by inositol phosphates. We endogenous protein kinases in rat liver supernatant could be by inositol phosphates. In these we were on the inositol phosphate-regulated over one the S with substrate Using this we identified a protein that was increased in its phosphorylation state by IP4, IP5, and IP6. This protein was identified as protein and was used in an to the inositol phosphate-regulated kinase. The in revealed that the of CK2 by and with the of the inositol phosphate-regulated protein kinase activity. evidence that CK2 catalytic activity could be regulated by inositol phosphates was by using expressed recombinant rCK2, which was to be regulated by inositol phosphates with a IP4 that was the as that observed for phosphorylation of protein in the S from rat of the most to from our studies was that CK2, contrary to established can exist in a state it has very low constitutive activity. In of liver supernatant from the first the phosphorylation of protein is low or in the of inositol phosphates is phosphorylation of protein This that in these CK2 has very low or activity. We that this may be to the of an endogenous in these that is able to inhibit basal CK2 activity. in the of the are inositol phosphates able to the catalytic activity of CK2, possibly by the This was supported by the fact that a fraction the could be used to the constitutive activity of recombinant in inositol phosphate to The identity of this has to be is however, that it is not one of the of the kinase of has high constitutive activity in the of the This is is evidence to the of the in of kinase activity as as substrate and CK2 J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, B. O. L.A. J. Biochem. PubMed Scopus Google Scholar). The not appear to be a the kinase were in phosphate to inhibit phosphatase activity and be to on the it is not a molecule that be in is to that CK2 with low constitutive activity is that the is This may an for the fact that this has not been studies are in to the identity of the inhibitory of CK2. process for CK2 have to the of this protein kinase and be to a of that the numerous influenced by CK2 phosphorylation. The involvement of inositol phosphates as a of CK2 many of the of a of cellular Of particular interest is the fact that IP4 is able to the activity of CK2. This inositol phosphate is the product of inositol 1,4,5-trisphosphate which IP3 to IP4 (2Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (528) Google Scholar). The in cellular of IP3 on the of the of is with a in the of IP4 R.A. S.R. J. PubMed Scopus Google Scholar, A.M. E.B. Mol. Google Scholar, R.A. S.R. J. PubMed Scopus Google Scholar). of the IP4 is by the inositol phosphatase that IP3, namely inositol P.W. F.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our that IP4, not IP3, can regulate the activity of CK2. This that CK2 could be under the regulated control of in IP4 and that this process may a potential novel signaling for IP4 a potential second messenger for the regulation of CK2, our that IP6 can regulate CK2 activity. In IP6 appears In contrast to IP4, IP6 is present in high concentrations in all eukaryotic cells (6Pittet D. Schlegel W. Lew D.P. Monod A. Mayr G.W. J. Biol. Chem. 1989; 264: 18489-18493Abstract Full Text PDF PubMed Google Scholar, 7Szwergold B.S. Graham R.A. Brown T.R. Biochem. Biophys. Res. Commun. 1987; 149: 874-881Crossref PubMed Scopus (111) Google Scholar), and these do not appear to cellular P.J. Michell R.H. 1993; PubMed Scopus Google Scholar). However, interest in IP6 as a potential molecule in cellular processes has been by studies that IP6 can with high affinity to a number of proteins involved in endo- and/or exocytosis (10Norris F.A. Ungewickell E. Majerus P.W. J. Biol. Chem. 1995; 270: 214-217Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 11Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 12Gaidarov I. Krupnick J.G. Falck J.R. Benovic J.L. Keen J.H. EMBO J. 1999; 18: 871-881Crossref PubMed Scopus (166) Google Scholar). In studies have a role for IP6 in the control of mRNA and DNA repair (4Shen X. Xiao H. Ranallo R. Wu W.H. Wu C. Science. 2003; 299: 112-114Crossref PubMed Scopus (291) Google Scholar, 5Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (313) Google Scholar, J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (443) Google Scholar, L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 16Hanakahi L.A. West S.C. EMBO J. 2002; 21: 2038-2044Crossref PubMed Scopus (86) Google Scholar). This has a of the cellular of IP6 in a array of (2Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (528) Google Scholar). Our that IP6 can regulate CK2 activity to this and that IP6 may have a signaling role in the direct control of protein phosphorylation. In this we identified protein as a substrate for CK2. However, the cellular function of protein is only identity with the rat phosphatidylcholine transfer protein, it is not protein has activity with transfer be to phosphatidylcholine transfer protein or transfer proteins are for CK2 and the phosphorylation of these proteins are regulated by inositol phosphates. In we the first evidence to suggest that CK2, being constitutively in a state with low basal activity. In this the kinase activity can be by inositol phosphates. In this phosphorylation may be regulated by a signaling process to second protein kinase We S. J. for and for with the cloning of protein.