Recombinant human collagens:characterization of type II collagen expressed in insect cells and production of types I-III collagen in the yeast <em>Pichia pastoris</em>

Abstract An efficient system for expressing recombinant human collagens is expected to have numerous scientific and medical applications, but this is difficult to achieve because most systems do not have sufficient levels of activity of prolyl 4-hydroxylase, the key enzyme of collagen synthesis. A recombinant form of human type II collagen, the main structural component of cartilage, was produced here in insect cells by coinfecting them with two baculoviruses, one coding for the proα chains of human type II procollagen, and the other for both the α and β subunits of human prolyl 4-hydroxylase. The amino acid composition of the recombinant form was very similar to that of the non-recombinant protein, with the exception that the hydroxylysine content was very low. The highest expression levels obtained in suspension cultures were 50 mg/l. An additional baculovirus coding for human lysyl hydroxylase was used to express type II collagen with a high hydroxylysine content. Marked differences in the rate of fibril formation in vitro and the morphology of the resulting fibrils were found between the recombinant type II collagens having 2 and 19 hydroxylysine residues/1000 amino acids, the maximal turbidity of the former being reached within 5 min, whereas the absorbance of the latter increased up to about 10 h. In addition, the latter collagen formed thin fibrils, whereas the former produced thick fibrils on a background of thin ones. The data indicate that regulation of the extent of lysine hydroxylation, and consequently of the amounts of hydroxylysine-linked carbohydrate units, may have major effects on collagen fibril formation. In order to study the expression of recombinant human collagens in yeasts, cDNAs for the proα chains of procollagens of type I, II and III were transformed into a recombinant P. pastoris strain expressing human prolyl 4-hydroxylase subunits. All the P. pastoris strains obtained produced full-length proα chains. Cells coexpressing the proα1(I) chains and prolyl 4-hydroxylase produced homotrimeric type I procollagen molecules, whereas cells coexpressing the proα1(I) and proα2(I) chains and prolyl 4-hydroxylase produced heterotrimeric molecules with the correct 2:1 chain ratio. pCα1(I) and pCα2(I) chains lacking the N propeptides assembled into pCcollagen molecules and yielded correctly folded and fully hydroxylated collagen molecules upon pepsinization. The Tm values of recombinant type I-III collagens produced in shaker flasks were about 38°C and the degree of hydroxylation of proline residues was lower than that in the corresponding non-recombinant collagens. When the recombinant collagens were produced in a 2-litre fermentor equipped with an O2 supply system, the expression levels increased markedly to 0.2–0.6 g/l. In addition, all these collagens were identical in 4-hydroxyproline content to the corresponding non-recombinant proteins, and all of them formed native-type fibrils

Täydennyskoulutustarpeet ja täydennyskoulutuksen merkitys uransa alussa olevien sosiaali-, terveys- ja kuntoutusalan ammattikorkeakouluopettajien osaamiselle

Abstract Up-to-date competences of educators in the social, health care and rehabilitation sector in universities of applied sciences are crucial for top-quality education. The purpose of this research was to describe the needs and meanings of continuing education for teachers starting their career. This study is a part of a national TerOpe project. The method of data collection was a theme interview. The interviews were arranged to four teacher groups consisting of 2—5 people. The people interviewed (n=14) were educators of nursing, fysiotherapy and social work on three universities of applied sciences located in different parts of Finland. The data were analysed with inductive content analysis. Those teachers who had less than five years of teacher experience wanted to have further education which answered to individual learning needs, were useful, included collegial interaction and were of high-quality pedagogically. Teachers also felt they need possibilities for development of their work organized by their employer, such as job orientation, supervision and mentoring. Teachers felt that they need to develop their own work, co-operation competence and they need personal professional development, and development of teaching competence and digital competence. The meaning of further education for teachers was diverse ways of gaining of professional knowledge and improvement on personal working abilities. The results can be used for the design and development of continuing education for social and health care teachers in the beginning of their career in universities of applied sciences.Tiivistelmä Sosiaali-, terveys- ja kuntoutusalan ammattikorkeakouluopettajien ajantasainen osaaminen on edellytys laadukkaalle koulutukselle. Tutkimuksen tarkoituksena oli kuvata uransa alussa olevien sosiaali-, terveys- ja kuntoutusalan opettajien täydennyskoulutustarpeita sekä täydennyskoulutuksen merkitystä heidän osaamiselleen. Tutkimus on osa valtakunnallista TerOpe hanketta. Aineistonkeruumenetelmänä oli teemahaastattelu. Haastattelut toteutettiin neljälle 2—5 hengen opettajaryhmälle. Haastateltavat (n=14) olivat hoitotyön, fysioterapian ja sosionomin koulutusohjelmien opettajia kolmesta eri ammattikorkeakoulusta eri puolilta Suomea. Aineisto analysoitiin induktiivisella sisällönanalyysilla. Alle viisi vuotta opettajana toimineet haastateltavat toivoivat tarvetta vastaavaa täydennyskoulutusta, joka on itselle hyödyllistä, sisältää kollegiaalista vuorovaikutusta ja on pedagogisesti laadukasta. Opettajat kokivat tarvitsevansa työnantajan tarjoamia työssä kehittymisen mahdollisuuksia kuten perehdytystä, työnohjausta ja mentorointia. Opettajana kehittymiseen he arvioivat tarvitsevansa oman työn ja yhteistyöosaamisen kehittämistä, henkilökohtaista ammatillista kasvua sekä opetusosaamisen ja digiosaamisen kehittymistä. Täydennyskoulutus merkitsi uransa alussa olevien opettajien osaamiselle ammattitaidon kehittymistä ja henkilökohtaisten työskentelyvalmiuksien vahvistumista. Tuloksia voidaan hyödyntää uransa alussa olevien ammattikorkeakouluopettajien ammatillisen kehittymisen ja täydennyskoulutuksen suunnittelussa ja kehittämisessä